How universal adhesive systems with nanoencapsulated flavonoids improve long-term bonding to caries-affected dentin

doi:10.21203/rs.3.rs-4974491/v1

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Research Article

How universal adhesive systems with nanoencapsulated flavonoids improve long-term bonding to caries-affected dentin

https://doi.org/10.21203/rs.3.rs-4974491/v1

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Objectives

To evaluate the effect of nano-encapsulated flavonoids in universal adhesives on resin-dentin bond-strength (µTBS) and nanoleakage (NL) on artificial caries-affected dentin (CAD).

Materials and Methods

Artificial CAD was created on the occlusal dentin surfaces of 60 human third molars by a microbiological assay. Nanoencapsulated quercetin (Q) and naringin (N) were incorporated into Prime&Bond Universal (PBU; Dentsply-Sirona) and Single Bond Universal (SBU, 3M ESPE). The adhesive systems with and without (control) flavonoids were applied to the CAD surface, which was validated by Knoop microhardness (KNH), and a 4-mm resin composite block (TPH Spectrum, Dentsply Sirona) was built up and light-cured. Specimens were sectioned into resin-dentin sticks and tested in tension in a universal testing machine. µTBS and NL tests were performed after 24-h and 6-month water storage (WS). The HL was subjected to micro-raman analysis to detect N and Q. µTBS and NL data were analyzed using a non-parametric three-way repeated measures ANOVA test followed by Bonferroni's test (α = 5%). KNH data was analyzed using a paired Student´s t test.

Results

CAD exhibited significantly lower KNH values than sound dentin. N and Q nanocapsules increased µTBS and reduced NL values after WS. At 24-h, PBU group showed higher NL values than SBU group, and the values decreased after WS. Q and N were detected within the HL.

Conclusions

Incorporating nanoencapsulated flavonoids may improve longevity of universal bonding systems applied to CAD.

Clinical Relevance:

Adhesives restorations with therapeutical compounds might be an option to create stable bonding over time.

dental adhesives

dentin bonding

caries-affected dentin

quercetin

naringin

collagen cross-linking

Nowadays, dental caries is a prevalent global chronic disease, affecting 60–90% of children and adults [1]. The dynamic caries process involves mineral loss due to endogenous bacteria that produce organic acid leading to variations in local pH values [1]. Alternating periods of tooth demineralization and remineralization due to pH drop causes caries lesions [2]. Furthermore, if the mineral loss continues, the porosity of inter-tubular dentin increases [3]. This enables the progression of the acids and bacteria deeper into dentin [4, 5], and creates two distinct layers: the outer layer, known as the caries-infected dentin, and the inner layer, known as the caries-affected dentin (CAD) [6, 7].

Commonly seen in dental practice, CAD represents a restorative challenge in terms of longevity and predictability, mainly due to its histological features [6, 7]. Nonetheless, resin composites restorations remain the most common direct procedure in restorative dentistry, which necessarily requires the use of dental adhesive systems. Therefore, longevity depends on how hybrid layer (HL) is preserved over time to guaranty a successful restoration [8]. In a realistic scenario, dental adhesive systems are prone to face these types of challenges, differently from traditional in vitro studies, which mainly report their mechanical properties on sound dentin [9].

Widely investigated in biomedical fields, flavonoids have shown to improve the stability and longevity of dentin bonding [10–12]. These molecules are natural polyphenols extracted from different plants, and their base structure consists of a fused benzo-γ-pyrone ring with a phenyl group. Hesperidin, proanthocyanidins, and epigallocatechin have shown therapeutic effects, reducing collagen degradation in adhesive restorations [13]. Furthermore, pretreatment solutions with flavonoids improves the bonding performance of universal adhesives in sound dentin For these reasons, the use of bioactive materials remain as a promising approach in dental restorative research.

Quercetin (Q) is a flavonoid belonging to the flavonol glycoside group, while Naringin (N) is part of the flavanone glycoside group. Several studies have demonstrated that Q and N present antibacterial, antioxidative and anti-inflammatory effects [14, 15]. When applied to CAD as primers, Q and N improved short- and long-term performance of universal bonding systems [16]. Despite such benefits, the additional step comprising primer application increases working time and the complexity of this approach. Consequently, clinicians may refuse to add this further step in the restorative procedure.

To avoid additional clinical steps, nanocapsules can be used as a reservoir when added to the adhesive system, promoting slow release of therapeutic substances into the targeted tissue [17, 18]. Thus, the effect of nanoencapsulated flavonoids incorporated in universal adhesives might be an innovative strategy to enhance bonding to CAD. Therefore, the aims of this in vitro study were: 1) to detect the presence of nanoencapsulated flavonoids within the HL by micro-Raman analysis; 2) to evaluate the resin-dentin bond-strength (µTBS) and NL on the bonding interface created by two universal adhesives, with nanoencapsulated flavonoid (N or Q), on CAD after 24h and following 6-month storage in distilled water (WS). Thus, the following null hypotheses were tested: a) the inclusion of nanoencapsulated flavonoids in two universal adhesives does not improve the 24h µTBS and NL values; b) the µTBS and NL values after 6-month water storage are not different from the 24h values; c) there is no difference in µTBS and NL values between brands.

Formulation of the experimental adhesives

Experimental adhesives were formulated using two universal adhesive systems: Prime&Bond Universal (PBUC; Dentsply-Sirona, Konstanz, Baden-Württemberg, Germany) and Single Bond Universal (SBUC; 3M ESPE, St Paul, MN, USA). Four experimental adhesives systems with nanoencapsulated PCL flavonoids, namely naringin (N) and quercetin (Q), were formulated, resulting in four experimental groups: PBUN, PBUQ, SBUN and SBUQ.

A mother solution was prepared by blending the nanoencapsulated and lyophilized flavonoids with the universal adhesives. The nanocapsules containing Q and N were weighed using an analytical balance (AW220, Shimadzu, Kyoto, Japan) and added to the adhesive solution using a micropipette (LABMATE 10 µl, PRLabor, São José, SC) and disposable tips. For every 100 µl of the mother solution, 900 µl of pure adhesive (SBU or PBU) was incorporated, resulting in a concentration of 1000 µg/ml [19]. The solution was mixed in Univortex mixer (Uniscience, SP, Brazil) for 2 min.

Experimental design and specimen preparation

The experimental design of the current study is displayed in Fig. 1. The in vitro study was previously approved by the local Ethical Committee (protocol number: 5.508.845). Sixty caries-free extracted human third molars were disinfected in 0.5% chloramine, stored in distilled water, and used within 3 months after extraction. The occlusal surfaces were wet ground to expose a flat, middle depth dentin surface with a 180-grit silicon carbide paper and were polished using 600-grit silicon carbide paper for 30 s to standardize the smear layer.

Simulated microbiological caries

Simulated microbiological caries was created as described in previous in vitro studies [16, 20]. Briefly, forty specimens were covered with epoxy resin (Araldite, Brascolo Ltda, São Bernardo do Campo, SP, Brazil) followed by a layer of nail varnish (Esmalte Risqué Natural Renda 8ML, Risqué, Liberdade, SP, Brazil), leaving the occlusal surface exposed. Each specimen was individually sterilized in a steam autoclave (Phoenix Ind. Brasileira, Araraquara, SP, Brazil) for 15 min at 121°C [21]. Then, the specimens were immersed in an 8-mL Falcon tube containing artificial caries solution composed of 9.25 g of brain heart infusion culture supplemented with 5.0 g of sucrose, 1.25 g of yeast extract, in 250 mL of distilled water and 100 µL of primary culture of S. mutans (ATCC 25175), with an approximate pH of 4.0, and were stored in an anaerobic jar (5% CO2) at 37°C for 48 h [21]. Each specimen was transferred to another 8-mL Falcon tube containing a new artificial caries solution and was stored for 14 days. The specimens were sterilized again as previously described and washed with distilled water.

In order to validate the simulated microbiological caries and CAD creation, twenty sound teeth were randomly selected for Knoop microhardness (KNH) test. The occlusal surfaces were wet ground to expose a flat dentin surface. Subsequently, they were sectioned in the middle, parallel to the long axis (Isomet 11/1180, Buehler Co, Lake Bluff, IL, USA), resulting in two halves. One half was subjected to 14-day induction of microbiological caries, as previously described [16, 22], while the dentin on other half remained sound and served as the control group. The occlusal dentin surfaces were polished with 600-grit silicon-carbide paper for 30 s in order to remove the caries-infected dentin and leave only CAD and a standardized smear layer on the occlusal surface

Bonding procedure

The surrounding enamel of all teeth was removed until the dentin surface was exposed with a diamond bur (#4137, KG Sorensen, Barueri, São Paulo, Brazil). Subsequently, adhesives were applied in the etch-and-rinse mode, following the manufacturer’s instructions (Table 1). A 4-mm thick resin composite (TPH Spectrum, Dentsply Sirona, Konstanz, Baden-Wurttemberg, Germany) block was incrementally built up. Each 2-mm composite layer was photo-cured for 20 s at 1200 mW/cm² (Radii Plus, SDI Limited, Victoria, Austrália). The irradiance was constantly checked with a radiometer (Bluephase meter II, Ivoclar Vivadent, Schaan, Liechtenstein). After 24-h storage in distilled water at 37°C, the restored teeth were sectioned into resin-dentin beams with a cross-sectional area of approximately 0.8 mm². Thirteen resin-dentin beams were evaluated after 24-h for µTBS, NL and micro-Raman analyses, while ten resin-dentin beams were stored in Eppendorfs containing 10mL of distilled water at 37°C for 6 months (WS) for µTBS and NL analyses. The storage solution was not replaced, and its pH was monitored monthly with pH indicator strips (pH 0–14 Universal Indicator, Merck KGaD, Darmstadt, Germany) to assure that its pH remained neutral.

Table 1

Composition and manufacturers’ instructions of the adhesive systems
Adhesives	Manufacturer	Composition^a	Aplicacation^b
Single Bond Universal (Batch Number: 2114800062)	3M ESPE, St Paul, MN, USA.	MDP phosphate monomer, dimethacrylate resins, HEMA, Vitrebond Copolymer, filler, ethanol, water, initiators, silane	1- Acid etch the surface with 37% phosphoric acid (Condac, FGM Prod. Odonto. Ltda, Joinville, SC, Brasil) for 15 s; 2- rinse the acid off for 30 s; 3- Air-blown dry te surface for 5 s; 3-. Apply actively the adhesive to the entire dentin surface with a microbrush for 20 s; 4- Direct a gentle stream of air over the liquid for about 5 s; 5. Light-cure for 10 s at 1200 mW/cm2 (Radii Plus, SDI Limited, Victoria, Austrália).
Prime&Bond Universal (Batch Number: 2203001010)	Dentsply Sirona, Konstanz, Baden-Wurttemberg, Germany	10-MDP, PENTA, bisacrylamide monomers, isopropanol, water, initiator, stabilizer	1- Acid etch the surface with 37% phosphoric acid (Condac, FGM Prod. Odonto. Ltda, Joinville, SC, Brazil) for 15 s; 2- rinse the acid off for 30 s; 3- Air-blown dry te surface for 5 s; 4. Apply actively the adhesive to the entire dentin surface with a microbrush for 20s; 5. Direct a gentle stream of air over the liquid for about 5 s; 6. Light cure for 10 seconds at 1200 m W/cm² (Radii Plus, SDI Limited, Victoria, Austrália).
^a MDP = methacryloyloxydecyl dihydrogen phosphate; HEMA = 2-hydroxyethyl methacrylate; PENTA = dipentaerythritol pentacrylate
^b As per manufacturers’ instructions

Validation of simulated microbiological caries

The adhesive interfaces created on sound and CAD dentin were polished under cooling water with 600 grit SiC paper for 30 s, followed by 1000, 1200, 2000 and 4000 grit SiC papers for 60 s each. The surface was subjected to a final polishing using a 0.25 µm diamond paste (Buehler Ltd., Lake Bluff, IL, USA) on a polishing cloth (Qprep Galaxy – ETA, QATM, Mammelzen, Germany). KNH was measured on the dentin surface 50 µm below the adhesive/dentin interface with a Knoop indenter (Shimadzu HMV II, Kyoto, Japan) with a static load of 25g for 30s. Each specimen received three indentations.

Microtensile bond strength (µTBS)

Fourteen resin-dentine beams from each tooth (n = 7) were attached to a Geraldeli jig [23] (Odeme Biotechnology, Joaçaba, SC, Brazil) with a cyanoacrylate resin (IC-Gel, bSi Inc., Atascadero, CA, USA) and were subjected to a tensile force on a universal testing machine (Kratos, Sao Paulo, SP, Brazil) at a crosshead speed of 0.5 mm/min. The failure mode was evaluated through an optical microscope (SZH-131, Olympus, Tokyo, Japan) at 40x and classified as cohesive in resin (failure within cohesive resin), cohesive in dentin (failure within cohesive dentin), adhesive (failure at resin-dentin interface), or mixed (failure at resin-dentin interface that included cohesive failure of the adjacent substrates). The premature failures were counted, recorded, and not included in the average of µTBS results.

Detection of flavonoids within the HL using micro-Raman spectroscopy

Three specimens from each tooth (n = 3) were selected and the adhesive interfaces were wet polished with 1500-, 2000-, and 2500-grit SiC paper for 15 s each. Then they were ultrasonically cleaned for 20 min in distilled water and stored in water for 24h at 37°C. Micro-Raman analysis (Senterra, Bruker Optik GmbH, Ettlingen, Baden-Wurttemberg, Germany) was performed on fifteen sites along the adhesive interface at 1-mm distance between each site. The adhesive layer, hybrid layer (HL), and dentin. were scanned using the computer-controlled x-y-z stage. The following micro-Raman parameters were set: 10 mW Neon laser with 785 nm wavelength, spatial resolution of ≈3 mm, spectral resolution ≈5 cm ^-1, and magnification of 100x (Olympus SZ40, London, UK) to a ≈1 mm beam diameter. Spectra were acquired with an accumulation time of 60s with 3 co-additions. After acquisition, the data was filtered for spikes and high frequency noises, and just spectra from the HL were considered.

Nanoleakage (NL)

Six specimens from each tooth (n = 6) were immersed in 50 wt% ammoniacal silver nitrate solution [24] in complete darkness for 24h, rinsed thoroughly in distilled water, and immersed in photo-developing solution for 8 h under a fluorescent light. Each specimen was polished with wet 600-, 1000-, 1200-, 1500-, 2000- and 2500-grit SiC paper followed by a 0.25 µm diamond paste (Buehler Ltd) using a polishing cloth (Qprep Galaxy – ETA, QATM, Mammelzen, Germany). Then, specimens were ultrasonically cleaned, air dried, mounted on stubs, dehydrated with colloidal silica for 24h and coated with gold sputter coated (MED 010, Balzers Union, Balzers, Liechtenstein). Resin–dentine interfaces were analyzed in a FEG-SEM operated in the backscattered mode at 12kV (VEGA 3 TESCAN, Shimadzu, Tokyo, Japan). Three micrographs of each resin–dentine bonded beam was obtained. The relative percentage of NL was measured using a public domain software (Image J, NIH, Frederick, MD, USA).

Statistical analysis

On observing non-normal distribution (Shapiro-Wilk test: W = 0.837, p = 0.001), the results for µTBS and NL were subjected to non-parametric three-way repeated ANOVA test followed by Bonferroni's test. KNH data were assessed for a normal distribution using the Shapiro-Wilk test and were compared using a paired Student´s t test. Statistical analyses were pre-set at significance level α = 5% using commercial statistical software (IBM SPSS Statistics 28, IBM Company, Armonk, New York, USA).

Simulated microbiological caries validation

The KNH values are presented in Table 2. CAD exhibited significantly lower hardness values than sound dentin (p<0.001).

Table 2

Knoop microhardness 50 µm below the adhesive/dentine interface
	Mean	Standard Deviation	p-value
Sound Dentin	65.6	5.50	<0.001
CAD	36.8	7.19	<0.001

Microtensile bond strength and fracture analysis

The µTBS values are presented in Table 3. The µTBS values decreased significantly over time in the C group (p<0.01), regardless of the adhesive, while the values in Q and N groups were significantly higher after WS than those observed at 24h interval (p < 0.001). No significant differences were observed among groups at 24h interval. Conversely, after WS, the Q and N groups showed higher µTBS values (p<0.01) than the C group. Q group showed the highest µTBS values after WS. The adhesive failure pattern was predominantly observed in all experimental groups and moments (Fig. 2.)

Table 3. µTBS (MPa) means (standard deviation) of control and experimental groups at two intervals.

	24h			WS
Group	PBU	SBU	Total	PBU	SBU	Total
C	23.9 (4.6)	21.5 (0.6)	22.7 (3.4)^Ba	19.1 (1.7)	18.5 (1.5)	18.8 (1.6)^Aa
N	22.6 (2.2)	23.9 (2.7)	23.3 (2.5)^Aa	26.9 (3.2)	24.6 (1.0)	25.7 (2.6)^Bb
Q	23.5 (1.4)	22.3 (1.9)	22.9 (1.7)^Aa	32.0 (2.7)	29.4 (3.0)	30.7 (3.1)^Bc

Means followed by same letter (uppercase letters within row; lowercase letters within column) are not significantly different (pre-set α=5%)

Detection of flavonoids within the HL using micro-Raman spectroscopy

The micro-Raman spectra from the HLs created using SBU and PBU are presented in Fig. 3. The white pointer indicates adhesive infiltration in all experimental groups at 1450cm^-1. SBUQ group presented a peak coalescence while PBUQ presented a peak shift resulting in higher wavenumbers at 1450 cm^-1. Figure 3A and 3C exhibits the characteristic N carbonyl peak at 1665 cm^-1, along with the ring bands, A and B rings at 1187 cm^-1 and the C ring at 1238 cm^-1. In Fig. 3B and 3D, Q exhibits the characteristic carbonyl peaks at 1611 cm^-1 and in the bending vibration of 638 cm^-1 in SBU specimens and 688 cm^-1 in PBU specimens. Besides, OH^- peaks were seen at 599 cm^-1 and 1183 cm^-1 in SBU specimens, as well as at 598 cm^-1 and 1365 cm^-1 in PBU specimens.

Nanoleakage analysis

The NL values are presented in Table 4 and representative images in Fig. 4. An increase in discontinuous silver deposits was identified at the bottom of the HL in the C group after WS (p < 0.001). Conversely, apparently, fewer voids were observed at the adhesive interfaces after WS when flavonoids were used. No significant differences were observed among groups at 24h. After WS, Q and N groups showed lower NL values than the C group (p<0.001). The specimens in the Q group showed the lowest NL values after WS (p<0.001).

PBU group showed significantly higher NL values at the 24h compared those after WS (p<0.016). No significant difference was observed between moments for the SBU adhesive. Moreover, the PBU group showed statistically higher NL values than the SBU group at 24h (p<0.009). There were no significant differences between PBU and SBU after WS.

Table 4. Nanoleakage (%) means (standard deviation) of control and experimental groups at two intervals.

	24-h			WS
Group	PBU	SBU	Total	PBU	SBU	Total
C	9.5 (2.7)	9.6 (4.8)	9.6 (3.9)^Aa	12.5 (3.4)	12.7 (3.0)	12.6 (3.2)^Bc
N	11.7 (3.5)	8.3 (3.2)	10.0 (3.7)^Ba	7.6 (2.0)	8.1 (2.8)	7.8 (2.4)^Ab
Q	9.9 (4.6)	8.0 (2.6)	8.9 (3.8)^Ba	5.1 (2.1)	6.2 (2.4)	5.6 (2.3)^Aa
Total	10.3 (3.8)^Bb	8.6 (3.7)^Aa		8.4 (4.0)^Aa	9.0 (3.8)^Aa

Means followed by the same letter (for group totals in columns: uppercase letters within row, lowercase letter within column; for adhesive totals in bottom row: uppercase letters between intervals for the total within the same adhesive, lowercase letters between the totals within the same interval) are not significantly different (pre-set α=5%)

Differently from previous studies [10, 16, 25], nanoencapsulated flavonoids were incorporated into the adhesive systems instead of being applied to the dentin surface beforehand. In this condition, after WS, the universal adhesives with nanoencapsulated flavonoids promoted higher µTBS values and lower NL values than the control group. In addition, the µTBS values in the control group decreased over time, while adhesives with nanoencapsulated flavonoids promoted higher µTBS values over time. Therefore, the first and second null hypotheses were rejected. Collagen cross-linking agents can improve biochemical and biomechanical properties, reducing biodegradation and preserving the long-term bonding stability in sound dentin and CAD [10, 11, 16]. Notably, the microhardness results are consistent with those from prior studies [26, 27], further validating that the current investigation was conducted on a CAD substrate.

In the current study, the micro-Raman spectra from the HL showed characteristic peaks of N and Q. Similarly, Krysa et al. (2022) reported a naringenin carbonyl peak at 1660 cm^− 1, A and B rings at 1191cm^− 1, C ring at 1235 cm^− 1; and Q carbonyl peak at 1615 cm^− 1 in stretching vibration and in bending vibrations 690 cm^− 1 and 642 cm^− 1, the OH peaks at out of plane bending 599 cm^− 1 and in plane bending at 1179 cm^− 1 and 1367 cm^− 1 [28]. It is worth noticing that the glycosidic form of naringenin is N, which presents similar bands as the spectra is based on the flavanone core [28].

Concerning the presence of Q within the HL, the SBUQ peak coalescence may be attributed to hydroxyl groups from Q and catechol moiety in the B-ring, as it has more conformers and reactivity than N [29]. On the other hand, the peak shift in PBUQ is likely associated with the presence of a double bond in C2 = C3 [30]. Therefore, these findings demonstrated that adhesives carried the polycaprolactone (PCL) nanocapsules into the demineralized dentin and open dentin tubules. As a consequence, the PCL nanocapsules acted as a transporter of Q and N into deeper areas within the demineralized intertubular dentin and dentinal tubules. PCL is an aliphatic polyester, which allows a controlled release of the active ingredient over a period that can last several days to weeks [17, 31]. As shown in the micro-Raman analysis, the presence of N and Q within the HL confirmed the slow and controlled release of flavonoids over time, contributing to the higher µTBS values after WS compared to the control group.

Exogenous cross-linking of the collagen matrix promoted by flavonoids provides an effective solution for preserving demineralized dentin, as shown in the current study and other studies [12, 16]. Flavonoids have antioxidant activities that inhibit the production of free radicals and scavenge oxygen-derived radicals [15]. Their antioxidant properties depend on their chemical structure: the phenolic hydrogens that enable them to donate molecules and the specific substitution patterns within the structure [32, 33]. Therefore, after WS, the exposed collagen at the bottom of the HL was strengthened by flavonoids, probably due to the high reactivity of their hydroxyl groups [14, 34].

When comparing the experimental groups, SBUQ and PBUQ groups exhibited higher µTBS values than the SBUN and PBUN groups after WS. Q crosslinks relies in four different types of forces: i) van der Waals forces, which allow interaction with the hydrophobic counterpart in other molecules [13], ii) electrostatic forces, increases surface-free energy [35], iii) hydrophobic forces, which promote the incorporation into the collagen structure [36] and iv) hydrogen bonds, allows the interaction with carbonyl groups of collagen fibrils [12]. Additionally, its hydroxyl functional groups at positions C-5 and C-3 have shown to be more efficient in inducing the electrophile responsive feature and displaying a higher antioxidant capacity [34, 37]. On the other hand, N possesses a phenolic ring C that is not conjugated due to C2 and C3 sp3 hybridization [38]. As a result, ring B is almost perpendicular to rings A and C, causing lower antioxidant capability [38]. Furthermore, N is moderately soluble in water, which causes steric hindrance of the scavenging group [39]. In this regard, one could state that flavonoids have proven antioxidant effects when applied to CAD.

Over time, SBU and PBU groups with Q and N presented lower NL values than the control group. This could be explained by the antioxidant effect of Q and N; due to their hydroxyl groups' high reactivity, flavonoids can donate hydrogen atoms, resulting in the formation of less reactive and more stable free radicals [34]. However, as previously mentioned, the antioxidant properties depend on the structural arrangement of functional groups as hydroxyl groups' configuration [40]. Based on their structure, Q belongs to the flavonol group, which presents a 3´, 4´-catechol structure in the B ring that enhances peroxidation, allowing the most effective scavenger of peroxyl, superoxide and peroxynitrite radicals [41]. On the other hand, N belongs to the flavone group, where its lack of catechol system on oxidation leads to the formation of unstable radicals with weak scavenging potential [14]. This aspect may explain the lower NL values in Q groups when compared to those observed in N groups after WS. Interestingly, the NL values from C, Q, and N groups have apparently impacted the µTBS values (Table 4). In other words, as the percentage of silver deposition within the HL increased, a corresponding decrease in µTBS values was observed in these groups. The gradual increase in silver penetration within the HL is attributed to water penetration through the NL channels, resulting in lower bond strengths [42].

Discontinuous silver deposits detected at the bottom of the HL at 24h are likely a result of unprotected ester linkage of the methacrylate-based materials that are susceptible to hydrolysis [43]. Conversely, apparently fewer voids were seen after WS when flavonoids were used. These results can be attributed to the ability of flavonoid molecules to modify water dynamics into dentin by reducing extracellular matrix water content and thereby improving adhesive infiltration [16, 25]. However, PBU showed a significant increase in 24h NL values when compared to SBU and a significant reduction after WS. This may be attributed to PBU composition, which includes isopropanol, a highly water-soluble solvent known to increase the adhesive susceptibility to early degradation [44]. Hence, the third null hypothesis was partially rejected.

Some limitations must be addressed in the present study. This study only evaluated the etch-and-rinse mode of universal adhesives. Thus, our findings should not be extrapolated to other adhesive systems. In addition, further effects of the use of polyphenols were not explored. For instance, current medical evidence has shown that these phytochemical compounds may stimulate physiological reactions to enhance the production of host anti-inflammatory and circulating substances, such as nitride oxide, to improve blood flow which may positively impact the protective effects of these compounds in a clinical scenario, showing multiple benefits beyond the chemical binding that creates a therapeutic effect in the collagen fibrils [14]. Further studies are required to address these issues.

Within the limitations of the current in vitro study, it is possible to conclude that the flavonoid nanocapsules:

Were able to penetrate the demineralized dentin along with the adhesive.

Were effective in reducing the effects of aging over the HL while avoiding HL degradation over time.

Improved the long-term µTBS values of universal bonding systems applied to CAD in etch-and-rinse mode.

Ethical approval

Approval was obtained from the local Ethical Committee of State University of Ponta Grossa (protocol number: 5.508.845).

Competing interests

The authors declare no competing interests.

Funding

This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) (grant number 001).

Author Contribution

C.F.P. contributed in conceptualization, investigation, methodology, visualization, writing – original draft. A.N. contributed in methodology, writing – review & editing. T.G.N. contributed in methodology, writing – review & editing. C.C.G.V. contributed in writing – review & editing. L.A.C. contributed in formal analysis, writing – review & editing. A.D.S contributed in conceptualization, investigation, validation, writing– review & editing, project Administrator. C.A.G.A. contributed in conceptualization, investigation, validation, writing– review & editing, supervision, project administrator.

Acknowledgement

AcknowledgementsBruna Hilgemberg is acknowledged for synthesizing the nanoencapsulated flavonoids.

Data availability

No datasets were generated or analysed during the current study.

Pitts NB, Zero DT, Marsh PD, Ekstrand K, Weintraub JA, Ramos-Gomez F, Tagami J, Twetman S, Tsakos G and Ismail A (2017) Dental caries. Nat Rev Dis Primers 3:17030. doi: 10.1038/nrdp.2017.30
Tabata M, Ratanaporncharoen C, Ishihara N, Masu K, Sriyudthsak M, Kitasako Y, Ikeda M, Tagami J and Miyahara Y (2021) Surface analysis of dental caries using a wireless pH sensor and Raman spectroscopy for chairside diagnosis. Talanta 235:122718. doi: 10.1016/j.talanta.2021.122718
Nakajima M, Kitasako Y, Okuda M, Foxton RM and Tagami J (2005) Elemental distributions and microtensile bond strength of the adhesive interface to normal and caries-affected dentin. J Biomed Mater Res B Appl Biomater 72:268-75. doi: 10.1002/jbm.b.30149
Isolan CP, Sarkis-Onofre R, Lima GS and Moraes RR (2018) Bonding to Sound and Caries-Affected Dentin: A Systematic Review and Meta-Analysis. J Adhes Dent 20:7-18. doi: 10.3290/j.jad.a39775
Mazzoni A, Tjaderhane L, Checchi V, Di Lenarda R, Salo T, Tay FR, Pashley DH and Breschi L (2015) Role of dentin MMPs in caries progression and bond stability. J Dent Res 94:241-51. doi: 10.1177/0022034514562833
Alves FB, Lenzi TL, Reis A, Loguercio AD, Carvalho TS and Raggio DP (2013) Bonding of simplified adhesive systems to caries-affected dentin of primary teeth. J Adhes Dent 15:439-45. doi: 10.3290/j.jad.a28880
Perdigão J, May KN, Jr., Wilder AD, Jr. and Lopes M (2000) The effect of depth of dentin demineralization on bond strengths and morphology of the hybrid layer. Oper Dent 25:186-94.
Betancourt DE, Baldion PA and Castellanos JE (2019) Resin-Dentin Bonding Interface: Mechanisms of Degradation and Strategies for Stabilization of the Hybrid Layer. Int J Biomater 2019:5268342. doi: https://doi.org/10.1155/2019/5268342
Marshall GW, Jr., Marshall SJ, Kinney JH and Balooch M (1997) The dentin substrate: structure and properties related to bonding. J Dent 25:441-58. doi: https://doi.org/10.1016/s0300-5712(96)00065-6
Davila-Sanchez A, Gutierrez MF, Bermudez JP, Mendez-Bauer L, Pulido C, Kiratzc F, Alegria-Acevedo LF, Farago PV, Loguercio AD, Sauro S and Arrais CAG (2021) Effects of Dentine Pretreatment Solutions Containing Flavonoids on the Resin Polymer-Dentine Interface Created Using a Modern Universal Adhesive. Polymers (Basel) 13. doi: https://doi.org/10.3390/polym13071145
Porto I, Rocha ABB, Ferreira IIS, de Barros BM, Avila EC, da Silva MC, de Oliveira MPS, Lobo T, Oliveira J, do Nascimento TG, de Freitas JMD and de Freitas JD (2021) Polyphenols and Brazilian red propolis incorporated into a total-etching adhesive system help in maintaining bonding durability. Heliyon 7:e06237. doi: https://doi.org/10.1016/j.heliyon.2021.e06237
Epasinghe DJ, Yiu CK, Burrow MF, Tsoi JK and Tay FR (2014) Effect of flavonoids on the mechanical properties of demineralised dentine. J Dent 42:1178-84. doi: https://doi.org/10.1016/j.jdent.2014.07.002
Hiraishi N, Sono R, Sofiqul I, Yiu C, Nakamura H, Otsuki M, Takatsuka T and Tagami J (2013) In vitro evaluation of plant-derived agents to preserve dentin collagen. Dent Mater 29:1048-54. doi: https://doi.org/10.1016/j.dental.2013.07.015
Kumar S and Pandey AK (2013) Chemistry and biological activities of flavonoids: an overview. ScientificWorldJournal 2013:162750. doi: https://doi.org/10.1155/2013/162750
Maleki SJ, Crespo JF and Cabanillas B (2019) Anti-inflammatory effects of flavonoids. Food Chem 299:125124. doi: https://doi.org/10.1016/j.foodchem.2019.125124
Davila-Sanchez A, Gutierrez MF, Bermudez JP, Mendez-Bauer ML, Hilgemberg B, Sauro S, Loguercio AD and Arrais CAG (2020) Influence of flavonoids on long-term bonding stability on caries-affected dentin. Dent Mater 36:1151-1160. doi: https://doi.org/10.1016/j.dental.2020.05.007
Grabnar PA and Kristl J (2011) The manufacturing techniques of drug-loaded polymeric nanoparticles from preformed polymers. J Microencapsul 28:323-35. doi: https://doi.org/10.3109/02652048.2011.569763
Genari B, Leitune VCB, Jornada DS, Aldrigui BR, Pohlmann AR, Guterres SS, Samuel SMW and Collares FM (2018) Effect on adhesion of a nanocapsules-loaded adhesive system. Braz Oral Res 32:e008. doi: https://doi.org/10.1590/1807-3107BOR-2018.vol32.0008
Hilgemberg B (2022) Análise das propriedades mecânicas da interface de união e resistência de união de adesivos contendo flavonoides. Universidade Estadual de Ponta Grossa
Gutierrez MF, Bermudez J, Davila-Sanchez A, Alegria-Acevedo LF, Mendez-Bauer L, Hernandez M, Astorga J, Reis A, Loguercio AD, Farago PV and Fernandez E (2019) Zinc oxide and copper nanoparticles addition in universal adhesive systems improve interface stability on caries-affected dentin. J Mech Behav Biomed Mater 100:103366. doi: https://doi.org/10.1016/j.jmbbm.2019.07.024
Marquezan M, Corrêa FN, Sanabe ME, Rodrigues Filho LE, Hebling J, Guedes-Pinto AC and Mendes FM (2009) Artificial methods of dentine caries induction: A hardness and morphological comparative study. Arch Oral Biol 54:1111-7. doi: 10.1016/j.archoralbio.2009.09.007
Gutiérrez MF, Alegría-acevedo LF, Núñez A, Méndez-Bauer L, Ñaupari-Villasante R, de Souza JJ, Buvinic S, Dávila-Sánchez A, Fernández E and Loguercio AD (2022) In vitro biological and adhesive properties of universal adhesive systems on sound and caries-affected dentine: 18 months. International Journal of Adhesion and Adhesives 114:103107. doi: https://doi.org/10.1016/j.ijadhadh.2022.103107
Perdigão J, Geraldeli S, Carmo AR and Dutra HR (2002) In vivo influence of residual moisture on microtensile bond strengths of one-bottle adhesives. J Esthet Restor Dent 14:31-8. doi: 10.1111/j.1708-8240.2002.tb00145.x
Tay FR, Pashley DH, Suh BI, Carvalho RM and Itthagarun A (2002) Single-step adhesives are permeable membranes. J Dent 30:371-82. doi: https://doi.org/10.1016/s0300-5712(02)00064-7
Leme-Kraus AA, Aydin B, Vidal CM, Phansalkar RM, Nam JW, McAlpine J, Pauli GF, Chen S and Bedran-Russo AK (2017) Biostability of the Proanthocyanidins-Dentin Complex and Adhesion Studies. J Dent Res 96:406-412. doi: https://doi.org/10.1177/0022034516680586
Ceballos L, Camejo DG, Victoria Fuentes M, Osorio R, Toledano M, Carvalho RM and Pashley DH (2003) Microtensile bond strength of total-etch and self-etching adhesives to caries-affected dentine. J Dent 31:469-77. doi: 10.1016/s0300-5712(03)00088-5
Joves GJ, Inoue G, Sadr A, Nikaido T and Tagami J (2014) Nanoindentation hardness of intertubular dentin in sound, demineralized and natural caries-affected dentin. Journal of the Mechanical Behavior of Biomedical Materials 32:39-45. doi: https://doi.org/10.1016/j.jmbbm.2013.12.017
Krysa M, Szymanska-Chargot M and Zdunek A (2022) FT-IR and FT-Raman fingerprints of flavonoids - A review. Food Chem 393:133430. doi: 10.1016/j.foodchem.2022.133430
Uyeki SC, Pacheco CM, Simeral ML and Hafner JH (2023) The Raman Active Vibrations of Flavone and Quercetin: The Impact of Conformers and Hydrogen Bonding on Fingerprint Modes. J Phys Chem A 127:1387-1394. doi: https://doi.org/10.1021/acs.jpca.2c06718
Daood U, Swee Heng C, Neo Chiew Lian J and Fawzy AS (2015) In vitro analysis of riboflavin-modified, experimental, two-step etch-and-rinse dentin adhesive: Fourier transform infrared spectroscopy and micro-Raman studies. Int J Oral Sci 7:110-24. doi: https://doi.org/10.1038/ijos.2014.49
Siracusa V, Rocculi P, Romani S and Rosa MD (2008) Biodegradable polymers for food packaging: a review. Trends in Food Science & Technology 19:634-643. doi: https://doi.org/10.1016/j.tifs.2008.07.003
Li X and Wang Q (2016) Analysis of the inherent instability of the interpolating moving least squares method when using improper polynomial bases. Engineering Analysis with Boundary Elements 73:21-34. doi: https://doi.org/10.1016/j.enganabound.2016.08.012
Chen G-L, Fan M-X, Wu J-L, Li N and Guo M-Q (2019) Antioxidant and anti-inflammatory properties of flavonoids from lotus plumule. Food Chemistry 277:706-712. doi: https://doi.org/10.1016/j.foodchem.2018.11.040
Mondal DS and Syed TR (2020) Flavonoids: A vital resource in healthcare and medicine (Includes their effective action on COVID-19). Pharmacology & Pharmacy 8:91-104. doi: https://doi.org/10.15406/ppij.2020.08.00285
Hass V, da Maceno Oliveira TB, Cardenas AFM, de Siqueira FSF, Bauer JR, Abuna G, Sinhoreti MAC, de Souza JJ and Loguercio AD (2021) Is it possible for a simultaneous biomodification during acid etching on naturally caries-affected dentin bonding? Clin Oral Investig 25:3543-3553. doi: 10.1007/s00784-020-03677-8
Fang M, Liu R, Xiao Y, Li F, Wang D, Hou R and Chen J (2012) Biomodification to dentin by a natural crosslinker improved the resin-dentin bonds. J Dent 40:458-66. doi: https://doi.org/10.1016/j.jdent.2012.02.008
Jurasekova Z, Domingo C, Garcia-Ramos JV and Sanchez-Cortes S (2014) Effect of pH on the chemical modification of quercetin and structurally related flavonoids characterized by optical (UV-visible and Raman) spectroscopy. Phys Chem Chem Phys 16:12802-11. doi: https://doi.org/10.1039/c4cp00864b
Celiz G, Suarez SA, Arias A, Molina J, Brondino CD and Doctorovich F (2019) Synthesis, structural elucidation and antiradical activity of a copper (II) naringenin complex. Biometals 32:595-610. doi: https://doi.org/10.1007/s10534-019-00187-3
Alam MA, Subhan N, Rahman MM, Uddin SJ, Reza HM and Sarker SD (2014) Effect of citrus flavonoids, naringin and naringenin, on metabolic syndrome and their mechanisms of action. Adv Nutr 5:404-17. doi: https://doi.org/10.3945/an.113.005603
Céliz G, Daz M and Audisio MC (2011) Antibacterial activity of naringin derivatives against pathogenic strains. J Appl Microbiol 111:731-8. doi: https://doi.org/10.1111/j.1365-2672.2011.05070.x
Pandey SK, Manogaran D, Manogaran S and Schaefer HF, 3rd (2017) Quantification of Hydrogen Bond Strength Based on Interaction Coordinates: A New Approach. J Phys Chem A 121:6090-6103. doi: https://doi.org/10.1021/acs.jpca.7b04752
Okuda M, Pereira PN, Nakajima M, Tagami J and Pashley DH (2002) Long-term durability of resin dentin interface: nanoleakage vs. microtensile bond strength. Oper Dent 27:289-96.
Cardoso MV, de Almeida Neves A, Mine A, Coutinho E, Van Landuyt K, De Munck J and Van Meerbeek B (2011) Current aspects on bonding effectiveness and stability in adhesive dentistry. Aust Dent J 56 Suppl 1:31-44. doi: https://doi.org/10.1111/j.1834-7819.2011.01294.x
Gutiérrez MF, Alegría-Acevedo LF, Méndez-Bauer L, Bermudez J, Dávila-Sánchez A, Buvinic S, Hernández-Moya N, Reis A, Loguercio AD, Farago PV, Martin J and Fernández E (2019) Biological, mechanical and adhesive properties of universal adhesives containing zinc and copper nanoparticles. Journal of Dentistry 82:45-55. doi: https://doi.org/10.1016/j.jdent.2019.01.012

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How universal adhesive systems with nanoencapsulated flavonoids improve long-term bonding to caries-affected dentin

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Abstract

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Materials and Methods

Results

Conclusions

Clinical Relevance:

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Introduction

Materials and methods

Formulation of the experimental adhesives

Experimental design and specimen preparation

Simulated microbiological caries

Bonding procedure

Validation of simulated microbiological caries

Microtensile bond strength (µTBS)

Detection of flavonoids within the HL using micro-Raman spectroscopy

Nanoleakage (NL)

Statistical analysis

Results

Simulated microbiological caries validation

Microtensile bond strength and fracture analysis

Detection of flavonoids within the HL using micro-Raman spectroscopy

Nanoleakage analysis

Discussion

Conclusions

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Funding

Author Contribution

Acknowledgement

Data availability

References

Additional Declarations

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