Influence of collagen cross-linkers addition in phosphoric acid on dentin biomodification and bonding of an etch-and-rinse adhesive

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Influence of collagen cross-linkers addition in phosphoric acid on dentin biomodification and bonding of an etch-and-rinse adhesive D.M. De-Paula a,b , D. Lomonaco a,c , A.M.P. Ponte b , K.E. Cordeiro b , M.M. Moreira a,b , S.E. Mazzetto c , V.P. Feitosa b,∗ a b c

Postgraduate Program in Dentistry, Federal University of Ceará, Fortaleza, Brazil Paulo Picanc¸o School of Dentistry, Fortaleza, Brazil Department of Organic and Inorganic Chemistry, Federal University of Ceará, Fortaleza, Brazil

a r t i c l e

i n f o

a b s t r a c t

Article history:

Objective. To investigate the effects of natural collagen cross-linkers incorporation in phos-

Available online xxx

phoric acid etchant on dentin biomodification, microtensile bond strength (␮TBS) and nanoleakage (NL) of a two-step etch-and-rinse adhesive.

Keywords:

Methods. Experimental aqueous solution of 37% ortho-phosphoric acid were prepared with

Dentin

the addition of 2% biomodification agents: Lignin (LIG) from industrial paper production

Lignin

residue, Cardanol (CARD) from cashew-nut shell liquid, and Proanthocyanidin (PAC) from

Cardanol

grape-seed extract. Negative control (NC) was acid solution without cross-linker whilst com-

Proanthocyanidin

mercial control (CC) was Condac 37 gel (FGM). Dentin specimens were assayed by FTIR after 15 s etching to detect collagen cross-linking. Extracted third molars were used for ␮TBS (n = 7) and fracture mode analysis of Optibond S (Kerr), tested after 24 h or 1000 thermal cycles. NL was surveyed by SEM. Statistical analysis was performed with two-way ANOVA and Tukey’s test (p < 0.05). Results. FTIR confirmed cross-linking for all agents. ␮TBS of CC was the highest (46.6 ± 6.2 MPa), but reduced significantly after aging (35.7 ± 5.2 MPa) (p < 0.001). LIG (30.6 ± 3.7 MPa) and CARD (28.3 ± 1.8 MPa) attained similar ␮TBS which were stable after aging (p > 0.05). Fracture mode was predominantly adhesive. At 24 h, all groups showed presence of silver uptake in hybrid layer, except CARD. After aging, CARD- and LIG-treated specimens exhibited little amount of silver penetration. CC, PAC and NC showed gaps, great nanoleakage at hybrid layer and presence of water channels in adhesive layer. Significance. Altogether, ortho-phosphoric acid incorporated with LIG and CARD promotes stable resin-dentin bond strength with minor nanoleakage after aging, thereby achieving therapeutic impact without additional clinical steps. © 2019 Published by Elsevier Inc. on behalf of The Academy of Dental Materials.

∗

Corresponding author at: Research Division, Paulo Picanc¸o School of Dentistry, 900 Joaquim Sá St., Fortaleza, 60135-218, Ceará, Brazil. E-mail address: [email protected] (V.P. Feitosa). https://doi.org/10.1016/j.dental.2019.11.019 0109-5641/© 2019 Published by Elsevier Inc. on behalf of The Academy of Dental Materials.

Please cite this article in press as: D.M. De-Paula, D. Lomonaco, A.M.P. Ponte et al.. Influence of collagen cross-linkers addition in phosphoric acid on dentin biomodification and bonding of an etch-and-rinse adhesive. Dent Mater (2019), https://doi.org/10.1016/j.dental.2019.11.019

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1.

Introduction

The threshold research regarding the effectiveness of acid etching dental hard tissues for the sake of improving bonding was the investigation of Buonocore [1]. In that occasion, 85% phosphoric acid was employed to bond acrylic resin to enamel. Later, Nakabayashi et al. [2] demonstrated bonding mechanism relies on homogeneous infiltration and in situ polymerization of monomers in etched dentin, theoretically accomplishing a mechanically stable interface, the so-called hybrid layer. Current dental bonding agents are classified as etch-andrinse or self-etch [3]. The former addresses 30–40% phosphoric acid to demineralize (5–8 ␮m) dentin, exposing collagen mesh for infiltration of monomers to ensure the adhesion of composite restorations [4]. The exposed dentin matrix is composed by type-I collagen (90%) and non-collagenous proteins, such as proteoglycans and matrix metalloproteinases (MMPs) surrounding the tridimensional structure of collagen [5,6]. The discrepancy between etching depth and adhesive resin infiltration yields unprotected collagen, which is rapidly degraded by activated MMPs. Also, acidic pH generated by bacteria metabolism may activate MMPs and induce release of cathepsins, which accelerate collagen degradation near hybrid layer [7]. In resin-dentin interface, rapid breakdown of unprotected collagen fibrils occurs mainly with etch-and-rinse adhesives due to the higher thickness of unprotected collagen, yielding restoration failures within the five initial years [4]. Water may also achieve polymer hydrolysis, thereby contributing for the bond short-lasting [8,9]. A feasible strategy to augment longevity of adhesive restorations is the biomodification of dentin substrate, promoting collagen cross-linking to increase biomechanical properties and diminishing biodegradation rates [10]. Such process reduces molecular mobility of unprotected collagen, inhibits MMPs and decreases water permeability [4,10,11]. The incorporation of biomodification agents in phosphoric acid etchant provides rapid therapeutic procedure without the need of an additional application of a biomodification primer. Proanthocyanidins (PAC) from grape-seed extract (Vitis vinifera) and chlorhexidine (CHX) added to the acid etchant previously demonstrated reliable outcomes on dentin bond stability [12–16]. Cardanol (CARD) from cashew nut shell liquid (Anacardium occidentale) is a product of discard from nut industry, which recently demonstrated optimal collagen cross-linking ability [17], improving mechanical properties and biodegradation resistance, without staining the dentin [18]. Also, lignin (LIG) is a natural polymer from plant cell walls, responsible for increasing their rigidity [19,20] with chemical structure rich in phenols (Fig. 1) potentially able to attain collagen cross-linking [10]. Both agents (CARD and LIG) were never investigated in Dentistry incorporated in phosphoric acid etchant in the attempt to improve the longevity of composite restorations. Indeed, their addition to the acid-etchant may increase user-friendly application of such therapeutic compounds. Therefore, the objective of this study was to evaluate the biomodification capacity of LIG and CARD in human dentin

added to phosphoric acid etchant and their role on dentin bonding of an etch-and-rinse adhesive. Hypotheses are (1) addition of all biomodification agents in experimental acids achieves collagen cross-linking, and (2) the incorporation of biomodification agents to etchant affords bond strength stability after aging, due to particular chemical structures with phenolic compounds (Fig.1)

2.

Materials and methods

2.1.

Specimens’ preparation

Thirty five sound human third molars were extracted for reasons apart from this investigation were used after approval of Institutional Ethics Committee (protocol 011133/2018). They were stored in 0.1% thymol solution and used within two months after extraction. Flat middle dentin surfaces were obtained after removing the occlusal enamel by cutting with a diamond saw adapted in a cutting machine (Cutmaster, São Paulo, Brazil). Exposed dentin surfaces were wet-abraded with 320-grit SiC polishing papers to obtain standardized smear layers.

2.2.

Experimental design

CARD (99% purity) was acquired from cashew nut shell liquid, which was gently donated by Amendoas do Brasil LTDA (Fortaleza, Brazil) separated by the methodology described by Lomonaco et al. [21]. Grape-seed extract (Vitis vinifera, >90% PAC, Meganatural Gold, Madera, USA) was used to attain PAC. Lignin (99% purity) was donated from paper industry Suzano Papel e Celulose SA (Limeira, Brazil) and used without further purification. Acid etchant solutions were prepared by adding 2 wt% biomodification agents [14] in 37% ortho-phosphoric acid solution of ethanol:water (at 1:1 ratio). Solutions’ pHs were standardized (<1.0) prior to using [16]. The experimental 37% phosphoric acid solution without biomodification agent was employed as negative control (NC). Commercial control (CC) was Condac 37% phosphoric acid gel (FGM Dental Products, Joinville, Brazil). Purification and characterization of cardanol and lignin are described in detail in supplemental material.

2.3.

FTIR spectroscopy

The dentin surfaces were examined after 15 s application of each one of the five acids and 30 s rinsing with distilled water. Spectroscopic analysis was attained by Fourier-transform infrared spectroscopy (Spectrum Frontier, Perkin Elmer Corp., Norwalk, USA) with 4 cm−1 resolution, 32 scans in transmittance mode. The spectra range was 1800−1000 cm-1 to survey peaks and shoulders at 1633 cm−1 (amide I), 1544 cm−1 (amide II), 1450 cm−1 (CH2 bending) and 1235 cm−1 (amide III) assigned to dentin collagen cross-linking, after normalization and baseline correction processes [22,23]. The analysis was realized in triplicate.

Please cite this article in press as: D.M. De-Paula, D. Lomonaco, A.M.P. Ponte et al.. Influence of collagen cross-linkers addition in phosphoric acid on dentin biomodification and bonding of an etch-and-rinse adhesive. Dent Mater (2019), https://doi.org/10.1016/j.dental.2019.11.019

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Fig. 1 – Chemical structures of biomodification agents surveyed. (A) Monolignols precursors of structural units forming the polymeric lignin molecule; (B) Chemical structure of purified cardanol; (C) Major structure of proanthocyanidins monomeric unit from grape seed extract.

2.4.

Restorative procedures

The dentin surfaces were etched with experimental or commercial acids for 15 s and rinsed with distilled water for 30 s. Following, the two-step etch-and-rinse adhesive Optibond S (Kerr, Orange, USA) was actively applied for 20 s, slightly airthinned for 3 s and light-cured for 20 s with LED unit DB-685 (1100 mW/cm2, Dabi Atlante, Ribeirão Preto, Brazil). Composite build-ups were constructed with 1mm-thick layers of micro-hybrid composite Opallis (FGM), which were individually light-cured for 20 s [13]. Bonded specimens were stored in distilled water for 24 h.

2.5. mode

Microtensile bond strength (TBS) and Fracture

Bonded teeth (n = 7) were longitudinally cut in 1mm-thick slices and turned 90◦ to perform further cuts and obtain resin-

dentin sticks with approximately 1mm2 cross-sectional area. Half of sticks per tooth were tested immediately and another half was tested after 1000 thermal cycles (30 s in 5 ◦ C and 30 s in 55 ◦ C with 10 s interval) (Huber-Mechatronik TC45SD, SD Mechatronik, Feldkirchen-Westerham, Germany) [24,25]. Microtensile test was performed by attaching the resin-dentin sticks in Geraldeli’s jigs with cyanoacrylate glue, adapted in a microtensile machine (OM-100, Odeme, Luzerna, Brazil) and tested in tensile until fracture with 0.5 mm/min crosshead speed and 500 N load cell. Before testing, each stick was measured with digital calliper (0.01 mm precision) to obtain the bond strength in megapascals (MPa) [13]. Pre-test fractures were not frequent (less than 3 per group) and were included as 0 MPa. All fractured sticks were examined in a stereomicroscope (60× magnification, Stereo-zoom S8, Leica, Heidelberg, Germany) in order to identify the failure modes, which were classified as adhesive, cohesive in dentin,

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cohesive in composite or mixed (partially adhesive and cohesive).

2.6.

Nanoleakage assessment

Two resin-dentin sticks from each subgroup were surveyed for interfacial silver nanoleakage following the protocol of Tay et al. [25], using 50% ammoniacal silver nitrate solution. Briefly, specimens were immersed in the tracer silver solution for 24 h protected from light, washed with distilled water and immersed in photodeveloping solution for 8 h under fluorescent light. Afterwards, they were embedded in epoxy resin and polished with SiC papers up to 4000-grit and 1-␮m diamond paste (Buehler, Coventry, UK) in polishing clothes [26]. They were cleaned for 5 min in ultrasonic bath after each polishing step and dehydrated for 24 h in an incubator with silica gel at 37 ◦ C. They were sputter coated with gold and observed in scanning electron microscopy (Quanta FEG, FEI, Amsterdam, Netherlands) in backscattered electron mode with 10 kV, 10 mm working distance and standardized 3000× magnification.

2.7.

Statistical analysis

Initially, data from ␮TBS (MPa) were subjected to Shapiro-Wilk test to evaluate the normal distribution. Then, they were subjected to two-way ANOVA (five levels of acids, and two levels of aging) and Tukey’s post-hoc test (˛ = 0.05).

3.

Fig. 2 – FTIR spectra of dentin specimens conditioned with different acids. Shoulders at ∼1400 cm−1 and ∼1080 cm-1 increased only with three biomodification agents. (A) Dentin etched with commercial control acid; (B) Dentin conditioned with negative control acid; (C) Specimens etched with lignin-containing acid; (D) Specimens conditioned with PAC-containing acid; D Dentin substrate treated with cardanol-containing phosphoric acid.

Results

FTIR spectra of etched specimens are shown in Fig. 2. All biomodification agents attained increase of aforementioned peaks (1633 cm−1 , 1544 cm−1 , 1450 cm−1 and 1235 cm−1 ), with notable enlarging of peak 1544 cm−1 only with specimens conditioned with acids containing PAC, CARD and LIG, thereby demonstrating collagen cross-linking. Yet, shoulders at ∼1400 cm-1 [22] and ∼1080 cm−1 [23] emerged with the presence of all three biomodification agents. The outcomes of ␮TBS are summarized in Fig. 3. Commercial control attained superior bond strength initially, but with significant drop after aging (p < 0.001). Such decrease occurred also with negative control (p = 0.02). LIG (30.6 ± 3.7 MPa) and CARD (28.3 ± 1.8 MPa) achieved similar initial ␮TBS (p = 0.881), which remained stable and resembling after aging (30.9 ± 4.2 and 28.9 ± 4.3 MPa respectively; p = 0.935). The comparison between treatments after aging demonstrated no statistical difference between commercial control, cardanol and lignin (p > 0.05). PAC-containing acid yielded the lowest initial bond strength (17.7 ± 0.9 MPa) with statistical drop after aging (9.9 ± 2.3 MPa; p = 0.003). The spreading of failure patterns is presented in Fig. 4. Adhesive fractures were predominant in most groups, particularly after aging. However, with commercial control acid at 24 h, cohesive in dentin was slightly higher than adhesive mode. Nanoleakage representative micrographs are depicted in Fig. 5. Striking silver infiltration was observed in interfaces of commercial control (at 24 h and after aging), LIG (24 h) and negative control (at 24 h and after aging). Interfacial gaps were

Fig. 3 – Means and standard deviations of resin–dentin microtensile bond strength (␮TBS) of all groups. Capital letters represent statistical differences in 24 h, whilst lowercase letters depict significant differences after aging. The black lines with p-values on the top indicate differences in the same group before and after aging. NC: Negative Control; CARD: Cardanol; PAC: Proanthocyanidin; LIG: Lignin; CC: Commercial Control.

frequent in negative control (at 24 h and after aging), and after aging with commercial control and with PAC-containing acid. LIG after aging and Cardanol demonstrated the highest resistance against silver impregnation, representing resin-dentin bonds almost devoid of defects and water.

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Fig. 4 – Failure mode percentages obtained in all groups after microtensile test. NC: Negative Control; CARD: Cardanol; PAC: Proanthocyanidin; LIG: Lignin; CC: Commercial Control.

4.

Discussion

The incorporation of collagen cross-linkers in phosphoric acid etchant is more feasible than their application as a therapeutic primer, which adds a further clinical step to the procedure. Indeed, the use of etch-and-rinse adhesives following dentin phosphoric acid etching is still widely employed. However, stable dentin bonds are accomplished especially by adding a biomodification agent to the acid etchant [12,13]. The thermocycling aging protocol addressed herein was similar to the regimen of Cotes et al. and El-Deeb et al. [24,25], which demonstrated ␮TBS reduction resembling to simulated pulpal pressure challenge [27]. As expected, experimental water/ethanol solutions of phosphoric acids achieved lower substrate etching than commercial acid gel. However, experimental acid containing biomodification agents attained collagen cross-linking (Fig. 2), resulting in acceptance of first hypothesis. Nevertheless, bonding stability was obtained only with cardanol and lignin, but not with PAC. Therefore, the second hypothesis is accepted for CARD and LIG, but rejected for PAC. Protection against degradation of demineralized dentin collagen, in presence of water, is currently clinically undertaken by means of MMP inhibitors in order to augment longevity of resin-dentin bonds [28]. Nonetheless, some investigations [12,14] recently demonstrated, by using ␮TBS and nanoleakage experiments, dentin bond stability of two-step etch-and-rinse adhesives when acid etchant was doped with collagen cross-linking agents, even after five years aging in distilled water [13]. Their findings corroborate with the present outcomes, once both acids free of biomodification agents (neg-

ative and commercial controls) did not protect hybrid layer (Fig. 5) and suffered bond strength decrease (Fig. 3) after aging, with consequent increase of adhesive failure percentage (Fig. 4). The presence of CARD and LIG in acid etchant promoted hybrid layer protection against hydrolysis and MMPs, as they maintained the bond strength stable with relatively high adhesive fracture pattern (Fig. 4). With these particular agents, few silver uptakes were observed in electron microscopies (Fig. 5). Concerning cardanol, its hydrophobic 15-carbon chain may impair the breakdown of the molecule in very acidic pH (<1). Nevertheless, the long time in acidic solution may likely achieve the breaking of double bond from alkene in the carbon chain. This might allow possible covalent bonds with collagen amino acids in addition to oligomerization of cardanol molecules, attaining higher degree of reticulation [29]. Moreover, cardanol is a relatively small molecule, which may rapidly penetrate around collagen fibrils [18,30,31] in the short application time used (15 s). At last, the hydroxyl in cardanol molecular may enhance collagen cross-linking by the formation of hydrogen bonds [17,18]. Due to its hydrophobic nature, high amount of aromatic rings and molecule complexity based on p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) (Fig. 1), lignin possess high resistance to acidic environments [32,33]. Besides, the irregular, branched and amorphous architecture of lignin with tridimensional bonds may accomplish a suitable arrangement between collagen fibrils, thereby protecting collagen mesh similarly to plant cell walls [20]. This explains optimal outcomes of lignin-containing acid in preserving bond strength (Fig. 3) and increasing the water resistance of resin-dentin interfaces (Fig. 5).

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Fig. 5 – SEM images of the bonded interfaces assayed at 24 h and after aging. The pointers represent water-trees into the adhesive layer; arrows highlight silver infiltration into the hybrid layer, and asterisks indicate gaps within the adhesive layer. In 24 h, all groups showed presence of silver in the HL, except CARD. After aging, CARD and LIG revealed little silver uptake. CC, PAC and NC depicted notable silver impregnation in HL, water-trees in the adhesive layers as well as interfacial gaps. NC: Negative Control; CARD: Cardanol; PAC: Proanthocyanidin; LIG: Lignin; CC: Commercial Control; C: composite resin; Ad: adhesive layer; HL: hybrid layer; D: dentin.

In the case of acid containing PAC from grape-seed extract, the initial bond strength was the lowest among all acids, with a further significant drop after aging (Fig. 3). Suitable explanations for this are the short application time (15 s), much lower than periods employed previously in literature, such as 10 min [11] and 1 h [34,35]. A similar negative result was found in the study of Moreira et al. [18], which observed severe collagen degradation for specimens treated with PAC, even with 1 min application as a primer. As well as, in study of two-year clinical evaluation of cervical restorations with PAC added to two-step etch-and-rinse adhesive [36]. Yet, the acidic media may promote the breakdown of B-type interflavane links of PAC [31,35,37], which is a condensed tannin. Such bonds are less stable in low pHs, thereby triggering the breaking of 3O-gallate esters [38]. Indeed, the absence of such links in the chemical structure of cardanol and lignin (Fig. 1) may afford more resistance in the acid etchant solution/gel. Moreover, cardanol and lignin possess overall more hydrophobic chemical feature in their chemical structure, thereby contributing to the stability of dentin bond and formation of hydrophobic interactions with collagen fibrils [17,18]. A further issue concerning proanthocyanidins from grapeseed extract is the phosphoric acid concentration (37%), which was based on that of commercial control for standardization purposes. However, this high acid content may lead to non-synchronized penetration of PACs, which are formed by dimeric, trimeric and oligomeric structures possessing different molecular weights. Indeed, this acidic environment with water and ethanol as solvents may afford formation of micelles, which certainly contributed negatively to the infiltration of PACs in collagen mesh at intertubular dentin. With control acids and PAC-containing one, demineralized collagen fibrils remained unprotected and prone to the activity of MMPs and other enzymes [16]. Liu et al. [16] suggests that the incorporation of PAC from grape-seed extract in phosphoric acid should be performed with acid concentrations lower than 20%. Another explanation for the lack of benefits with PAC-containing acid relies on the concentration of PAC. We standardized biomodification agent concentration in 2% according to Loguercio et al. [14], whilst most investigations employ PAC in 6.5% [11,18,34,35,39]. Indeed, this reduction in PAC concentration may hinder its efficacy as biomodification agent. Cardanol and lignin are products with sustainability appeal, once they are industrial wastes. Annual production of cashew nut shell liquid (with cardanol as major component) is 1000,000 tonnes [29]. In the case of lignin, the global production might be much higher, as it is obtained from industrial production of paper and cellulose. Based on the present study, further useful applications are demonstrated for both compounds, thereby generating a finality with higher added value.

5.

Conclusion

Phosphoric acid etchants incorporated with lignin and cardanol provide stable dentin bonds with low nanoleakage after aging and optimal collagen cross-linking, highlighting feasible usage in restorative dentistry.

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Acknowledgments This research was supported by Brazilian Ministry of Education and Science in part by the Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001 and grant 23038.006958/2014-96, FUNCAP, and CNPq grants 407291/2018-0 and 457931/2014-0.

Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/ j.dental.2019.11.019.

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Please cite this article in press as: D.M. De-Paula, D. Lomonaco, A.M.P. Ponte et al.. Influence of collagen cross-linkers addition in phosphoric acid on dentin biomodification and bonding of an etch-and-rinse adhesive. Dent Mater (2019), https://doi.org/10.1016/j.dental.2019.11.019