Isocyanate-terminated urethane-based dental adhesive bridges dentinal matrix collagen with adhesive resin

Accepted Manuscript Isocyanate-terminated urethane-based dental adhesive bridges dentinal matrix collagen with adhesive resin Rongchen Xu, Fan Yu, Li Huang, Wei Zhou, Yan Wang, Fu Wang, Xiang Sun, Gang Chang, Ming Fang, Ling Zhang, Fang Li, Franklin Tay, Lina Niu, Jihua Chen PII: DOI: Reference:

S1742-7061(18)30661-5 https://doi.org/10.1016/j.actbio.2018.11.007 ACTBIO 5757

To appear in:

Acta Biomaterialia

Received Date: Revised Date: Accepted Date:

10 July 2018 28 October 2018 5 November 2018

Please cite this article as: Xu, R., Yu, F., Huang, L., Zhou, W., Wang, Y., Wang, F., Sun, X., Chang, G., Fang, M., Zhang, L., Li, F., Tay, F., Niu, L., Chen, J., Isocyanate-terminated urethane-based dental adhesive bridges dentinal matrix collagen with adhesive resin, Acta Biomaterialia (2018), doi: https://doi.org/10.1016/j.actbio.2018.11.007

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Isocyanate-terminated urethane-based dental adhesive bridges dentinal matrix collagen with adhesive resin

Rongchen Xu1,†, Fan Yu1,†, Li Huang2,†, Wei Zhou1, Yan Wang1, Fu Wang1, Xiang Sun1, Gang Chang1, Ming Fang1, Ling Zhang1, Fang Li1, Franklin Tay1,3, Lina Niu1,3,*, Jihua Chen1,3,* 1State

Key Laboratory of Military Stomatology & National Clinical Research Center for Oral

Diseases & Shaanxi Key Laboratory of Stomatology, Department of Prosthodontics, School of Stomatology, The Fourth Military Medical University, Xi’an, China. 2State

Key Laboratory of Military Stomatology &National Clinical Research Center for Oral

Diseases & Shaanxi International Joint Research Center for Oral Diseases, Department of General Dentistry and Emergency, School of Stomatology, The Fourth Military Medical University, Xi’an, China. 3The

Dental College of Georgia, Augusta University, Augusta, Georgia 30912, USA.

† these

authors contributed equally to this work.

*Corresponding author: E-mail: [email protected](L.N.); [email protected](J.C.) KEYWORDS: adhesive; collagen; dentin bonding; isocyanate; urethane-based

1

Abstract Commercially available dental adhesives fail to chemically unite the demineralized collagen matrix with resinous materials within the resin-dentin interface. Sub-micron separations between the collagen fibrils and polymerized resin provide the backdrop for bond deterioration. Here, novel isocyanate-terminated urethane methacrylate precursors (UMP) were synthesized with the capacity to bond chemically to dentin collagen via covalent and hydrogen bonds. Collagen grafted with UMP also copolymerized with other methacrylate resin monomers, thereby producing a monoblock of chemically-linked biocomposite. The viscosity, degree of conversion and biocompatibility of UMP are comparable with commercially available resin monomers. An experimental adhesive containing 40% UMP demonstrated co-polymerization capability, good infiltration capacity and achieved higher immediate bond strength to dentin than the control commercially available adhesive. Improvement of dentin bonding by incorporation of UMP into dentin adhesives justifies future evaluation of the potential of these UMP-based adhesives in extending the longevity of resin-dentin bonds.

2

1. Introduction Resin-bonded restorations have become an indispensable treatment modality in contemporary restorative dentistry. The success of these restorations relies on the use of adhesive technology for bonding of these plastic materials to tooth structures. The etch-and-rinse and selfetch techniques are two different bonding strategies that are presently employed for bonding enamel and dentin [1]. For enamel, durable bonding may be achieved through a microinterlocking mechanism. Regardless of the strategy employed, dentin bonding relies on the formation of a hybrid layer, a structure consisting of both organic and inorganic substrate components reinforced by a polymerized resin matrix. Resin-dentin bonds are less durable than resin-enamel bonds because of the high collagen content that remains after etching dentin with acids in the case of etch-and-rinse adhesives, and functionalized acidic resin monomers in the case of self-etch adhesives [2]. These imperfectly-encapsulated collagen fibrils are vulnerable to time-dependent hydrolytic degradation by endogenous and exogenous collagenolytic enzymes, subsequently leading to bond deterioration with aging [3-6]. Two bonding mechanisms are responsible for the immediate bond strengths of contemporary adhesives: mechanical and chemical bonding. Because of the inherent limitation in demineralizing and infiltrating to the same depth, the etch-and-rinse approach produces imperfect micromechanical bonding, leaving a considerable amount of non-encapsulated collagen at the base of the hybrid layer, which results in suboptimal interlocking [3, 7]. Wet bonding helps maintain the integrity of the interfibrillar spaces, enabling better resin monomer penetration. When these resin monomers are in close contact with the demineralized collagen matrix, potential adhesive interactions occur via van der Waals, electrostatic or hydrogen bonding interactions [8]. However, these intermolecular interactions are complex and are not

3

completely understood [9-12]. In contrast, self-etch adhesives partially dissolve the mineral phase in the smear layer and superficial intact dentin to obtain micro-mechanical retention [13,14]. Some functional acidic resin monomers purportedly form chemical bonds with partiallydissolved carbonated apatite [15-17]. Inability of these functionalized acid resin monomers to bond chemically with collagen undermines the bond quality of self-etch adhesives [13, 18]. The purpose of acid etching in dentin bonding is to expose the collagen network and create space for the infiltration of adhesive resin monomers. Denuded collagen fibrils that are not completed protected by resin are vulnerable to degradation and compromise long-term bond stability [3]. Stable chemical interaction between functional resin monomers and the exposed collagen matrix may contribute to bond durability [13, 19]. Over the years, different strategies have been proposed for promoting interfacial stability via protection of the vulnerable demineralized collagen matrix. These strategies include inhibition of collagenolytic enzymes, collagen remineralization and collagen cross-linking [6, 20]. Among these strategies, chemical cross-linking approaches may be perceived as an imperfect form of chemical bonding. Chemical agents such as glutaraldehyde, carbodiimide, proanthocyanidin, hesperidin, and riboflavin have been tested for enhancing the biomechanical properties and biological stability of the collagen matrix [21]. Although the results are promising, problems remain to be solved before such practices become clinically acceptable [22]. However, these agents cannot integrate collagen with adhesive resin; they simply reinforce the collagen structure by cross-linking and may not be sufficient for durable bonding. Polymers containing a diisocyanate group, such as hexamethylene diisocyanate (HMDI), have been used as cross-linking agents for collagen-based biomaterials to minimize mechanical strains and control exogenous biodegradation [23]. Diisocyanate polymeric cross-linking agents 4

have demonstrated promising results in clinical applications such as reconstruction of the abdominal wall [24], mesentery [25], eyelid [26] and the chest wall [27]. The diisocyanate group has great affinity for collagen via covalent bonding with its -NH2 groups [28]. Based on this property, the authors opined that polymers with isocyanate (-NCO) groups may have novel applications in dentin bonding. By forming covalent bonds with the amino and hydroxyl groups of the collagen molecule [29], these polymers may be grafted to a demineralized collagen matrix. When a polymerizable methacrylate group is further introduced into these polymers, the methacrylate-functionalized isocyanate polymer will co-polymerize with other methacrylate resin monomers in the adhesive, thereby bridging the resin matrix and collagen to form a monoblock. Accordingly, isocyanate-terminated urethane methacrylate precursors (UMP) were synthesized and characterized to test the hypothesis that UMP is capable of cross-linking with demineralized dentin. An experimental adhesive was also formulated for testing the null hypothesis that there is no difference between the experimental adhesive and a commercially available two-step etch-and-rinse adhesive in their immediate bonding efficacy to acid-etched dentin. 2. Materials and methods 2.1 Reagents Isophorone diisocyanate (IPDI), dibutyltin dilaurate (DBTBL), di-n-butylamine (DNBA), bromocresol green (BCG), and methyl red (MR) were purchased from J&K Scientific (Beijing, China). 2-hydroxyethylmethacrylate (HEMA, 99%) was obtained from Aladdin (Shanghai, China). Bisphenol-A glycol dimethacrylate (Bis-GMA), triethylene glycol dimethacrylate (TEGDMA), diurethane dimethacrylate (UDMA), bisphenol-A ethoxylated dimethacrylate (Bis5

EMA),

choroform-d

(CDCl3),

camphorquinone

(CQ,

97%),

and

ethyl

4-(di-

methylamino)benzoate (EDMAB, 99%) were purchased from MilliporeSigma (St. Louis, MI, USA). Calcium sulfate (CaSO4), acetone, and dichloromethane were obtained from Hushi (Shanghai, China). 2.2 Synthesis and characterization of UMP The reaction scheme is illustrated in Fig. 1. One mole IPDI was added to a 100 mL flask and continuously purged with dry argon to remove oxygen and moisture. Then, 1 mole of HEMA was added dropwise to the flask. The catalysts consisted of DBTBL (0.5 mL) diluted in 10 mL acetone (purified with calcium sulfate by vacuum distillation). The reaction temperature was maintained at 40 ºC for 25 min with magnetic stirring. After the mixture was vigorously stirred, the hydrochloric acid-dibutyl amine titration method was used to determine the wt% of NCO. Absorption peaks were constantly monitored by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR; Shimadzu, FTIR-8400S, Kyoto, Japan). After 25 min of reaction, UMP containing both mono-substituted IPDI and di-substituted IPDI was generated and the total concentration of -NCO was 20 wt%. The UMP was further characterized by ATR-FTIR, 1H nuclear magnetic resonance spectrometry (ASCENDTM 600, Bruker, Rheinstetten, Germany) and high resolution mass spectrometry (Vion IMS QTof, Waters Corp., Philadelphia, PA, USA). One microliter of UMP was used for ATR-FTIR at room temperature and spectra were obtained with a resolution of 4 cm−1 in the range of 500–4000 cm−1. Proton NMR spectrum of UMP (deuterated CDCl3 as solvent) was recorded at 600 MHz with probe (PA BBO 600S3 BBF-H-D-05 Z SP). Moreover, UMP was dissolved into dichloromethane to 5 ppm and the m/z of each UMP component was analyzed with high resolution mass spectrometry (range: 50-2000 m/z, 100 ºC). Characterization 6

information for HEMA, IPDI with ATR-FTIR are summarized in Fig.2. 1HNMR, 13CNMR and HRMS data are included in the Supplementary Information (Suppl.-Fig.1, Suppl.-Fig.2 and Suppl.-Fig3). 2.2.1

Viscosity The viscosity of UMP and other dental monomers was measured with a LVDV-1 rotating

spindle viscometer (Fangrui, Shanghai, China) at 25 ºC in Pascal-seconds (Pa-s). One milliliter of sample was added to the plate and then mounted in the viscometer. The spindle of the viscometer was set at 0.5, 2 or 50 rpm to adjust the torque % to 45%–55% at a fixed speed. The actual viscosity was then recorded (n = 6). 2.2.2

Degree of conversion (DC)

To evaluate the DC by ATR-FTIR, 0.5 wt % CQ and 0.5 wt % EDMAB were mixed with the UMP. Methacrylate resin monomers Bis-GMA, TEGDMA, UDMA and HEMA containing the same concentration of photoinitiator and accelerator were used as controls. A thin layer of adhesive (5 mm diameter, 0.25 mm thick) was applied on the crystal of ATR-FTIR device with the help of an attached adhesive tape hole (5 mm diameter, 0.25 mm thick) [30, 31]. Prior to curing, the specimens were evaluated with a wavelength of 500–4000 cm-1, a resolution of 4 cm−1 and 32 scans to acquire the reference peak (1710 cm-1, C=O) of UMP, TEGDMA, UDMA, and HEMA and the reference peak of Bis-GMA (1608 cm-1, aromatic C=C). The aliphatic C=C (1638 cm-1) reaction peak was used for all groups. The specimens were subsequently cured with a light-emitting diode curing unit (Elipar S10, 3M ESPE, St. Paul, MN, USA) for 100 sec at 5 mm with an output intensity of 600 mW/cm2 and the absorbance of these peaks was recorded (n = 4). The DC of UMP, TEGDMA, UDMA and HEMA was calculated using the formula: DC (%) = [(A1638 cm-1/A1710 cm-1)uncured - (A1638 cm-1/A1710 cm-1)cured]/(A1638 cm-1/A1710 cm-1)uncured 7

x 100(%). The DC for Bis-GMA was calculated using the formula: DC (%) =[(A1638 cm1/A1608

cm-1)uncured - (A1638 cm-1/A1608 cm-1)cured] /(A1638 cm-1/A1608 cm-1)uncured x 100(%)

2.3 Cytotoxicity Human pulp stem cells (hDPSCs) were isolated and characterized in the authors’ previous study with the approval of the Institutional Review Board of the Stomatological Hospital, Fourth Military Medical University (FMMU; approval number: IRB-REV-2015036) [32]. Cells in the fifth passage were used to evaluate the cytotoxicity of UMP using a Cell Counting Kit-8 (CCK-8, 7-Sea Biotech, Shanghai, China). The cells were seeded in a 96-well plate at the concentration of 1×104 per well. The culture medium consisted of α-MEM supplemented with 20% fetal bovine serum (HyClone, Logan, UT, USA), 100 units/mL penicillin G, 100 mg/mL streptomycin and 50 mg/mL ascorbic acid (MilliporeSigma) at 37 °C in 5% CO2. After culturing for 24 h to enable cell adhesion, the culture medium was replaced with serum-free medium for 24 h starving. The cells were subsequently cultured with UMP and commercially available Bis-GMA, Bis-EMA, TEGDMA and UDMA. The resin monomers were dissolved in dimethyl sulfoxide and mixed with culture medium to obtain gradient concentrations (20, 40, 60, 80, 100, 120, 140, 160 and 180 μg/mL). Cells cultured without resin monomers served as the blank control. After incubation for 24 h, 10 μL of CCK-8 solution was applied per well and incubated for 4 h according to the manufacturer’s instructions. Cell viability was calculated by measuring the absorbance at 450 nm using a multi-plate reader (BIO-TEK, Winooski, VT, USA). The experiments were conducted in sextuplicate. Cell viability in the blank control group was normalized to 100%. Cell viability of the experimental groups was expressed as a percentage of the normalized blank control. 2.6 Interaction between demineralized dentin collagen matrix and UMP

8

Twenty-two intact, non-carious human third molars were collected from the Department of Oral Surgery of the School of Stomatology, FMMU, with the approval of the FMMU Institutional Review Board. The teeth were stored in 5% chloramine-T solution at 4 °C and used within 1 month after extraction. Ten molars were used for this experiment. The teeth were cut parallel to the occlusal surface, between the occlusal and the middle third (1 mm below the dentinoenamel junction) and sectioned into dentin discs (10 mm diameter, 1 mm thick) using a water-cooled diamondimpregnated cutting machine (SYJ-150A, Kejing, Shenyang, China). Two discs from the same tooth were randomly divided into a moist group and a dry group and used for ATR-FTIR observation. The dentin surface was polished with 600-grit silicon carbide paper and immersed in 10% phosphoric acid solution for 24 h to completely demineralize the mineralized dentin. The end-point of demineralization was monitored with ATR-FTIR; disappearance of the PO43− peak at 1004 cm-1 was indicative of complete demineralization. After rinsing with deionized water, the specimens were randomly divided into two groups (n = 10). Specimens in the first group were gently dried with filter paper to retain moisture; the other specimens were completely dried in an oven at 37 ºC for 24 h. Specimens from both groups were then immersed in UMP-acetone solution (volume ratio: 2/1.2) at 37 ºC for 5 min. The specimens were rinsed with acetone to remove unreacted UMP and measured with ATR-FTIR after the acetone became completely volatilized. 2.7 Adhesive formulation and tooth preparation An experimental two-step etch-and-rinse adhesive (EXP) containing UMP (40 wt%) was formulated to evaluate its bonding potential. According to the results of viscosity and molecular structure, UMP may be regarded as both a diluent co-monomer and a functional monomer. The 9

adhesive system also contained Bis-EMA (40 wt%) as a matrix monomer and acetone (20 wt%) as the solvent. The photoinitiator system consisted of 0.5 wt% CQ as the photoinitiator and 0.5 wt% EDMAB as the tertiary amine accelerator. A commercial two step etch-and-rinse adhesive, Single Bond 2 (SB2; 3M ESPE), served as the positive control. Twelve molars were used for the following experiments. The crown enamel was removed to expose the mid-coronal dentin. The latter was wet-polished with 1200-grit silicon carbide papers for 60 s with water cooling to create a standardized smear layer. The dentin was etched with 37% phosphoric acid gel (3M ESPE) for 15 s, rinsed with water for 30 s, and blotted dry with a cotton pellet, leaving a moist surface for adhesive application in accordance with the wet-bonding technique. Four prepared teeth were used immediately for contact angle evaluation. Four teeth were used for microtensile bond strength testing and the remaining four were used for morphological examination. Single Bond 2 was applied to the dentin surface according to the manufacturers’ instructions. Briefly, two consecutive coats of SB2 were applied to the dentin surface for 15 s with a fully saturated applicator. The adhesive saturated dentin was gently air-thinned for 5 s to evaporate the solvent and light-cured at room temperature using a light-emitting diode curing unit for 10 s with an output intensity of 600 mW/cm2. For the experimental adhesive, two consecutive coats of adhesive were applied for 10 s with an applicator, gently air-thinned for 5 s to evaporate the solvent. After carefully removing the visible air bubbles trapped in the adhesives, the adhesive-coated dentin was light-cured for 10 s with an output intensity of 600 mW/cm2. 2.8 Contact angle The contact angles of the adhesives on prepared dentin discs (10 mm diameter, 1 mm thick) were measured with a contact angle goniometer (EASY DROP K100, KRUSS Co., Hamburg, 10

Germany). The tip of a syringe was set to drop approximately 0.5 μL of EXP or SB2 onto the dentin surface each time, and the static angle of contact at the interface was traced. For each disc, photographs were recorded for 3 times within a time interval of 1s. At each time point, the right and left contact angles were measured and averaged. The water-contact angle on dentin when adhesives were completely polymerized was also evaluated. The adhesives were applied and polymerized in the manner previously described and a 0.5 μL droplet of deionized water was dispensed on each surface for recording the contact angle (n = 9). 2.9 Degree of conversion of adhesive The degree of conversion of EXP and SB2 was measured using ATR-FTIR as mentioned in the DC measurement. The reference (1608 cm-1, aromatic C=C) and reaction (1638 cm-1, aliphatic C=C) peaks of the adhesives were recorded. The degree of conversion was calculated using the formula: DC (%) = [1－ (1638 cm-1/1608 cm-1)cured/(1638 cm-1/1608 cm-1)uncured] x 100(%). Calculation of the degree of conversion for EXP was delayed for 3 min after the acetone was completely evaporated [30]. 2.10 Microtensile bond strength and fracture analysis Bonding was performed as previously described. After adhesive placement, composite build-up was performed using a hybrid resin composite (Filtek Z250, 3M ESPE) in four 1-mm increments. All the specimens were stored in deionized water at 37 °C for 24 h and sectioned in the mesiodistal and buccolingual directions across the bonded interface to obtain resin–dentin sticks with a cross-sectional area of 0.8 mm2–1.2 mm2 (n = 10). Testing was performed with a microtensile testing machine (EZ-TEST 500N, Shimadzu) at a crosshead speed of 1 mm/min. Debonded fragments were retrieved to determine the failure mode using a stereomicroscope (MLC-150, Motic, Decatur, GA, USA). Failure modes were classified as adhesive failure (A), 11

cohesive failure within dentin (D), cohesive failure within resin composite (C) and mixed failure that involved both interfacial and cohesive fracture (M). 2.11 Field emission-scanning electron microscopy (FE-SEM) The bonding interface was examined using FE-SEM (S-4800, Hitachi, Tokyo, Japan). After storing in distilled water at 37 ºC for 24 h, the bonded specimens were sectioned perpendicular to the bonded interface to produce 1-mm thick discs. The disc surfaces were treated with 37% phosphoric acid for 2 min to completely remove any smear layer created by cutting. The specimens were subsequently immersed in 5% sodium hypochlorite for 2 min to identify adhesive infiltration into dentin [33]. After ultrasonic irrigation for 5 min, the treated dentin discs were sputter-coated with gold and examined with FE-SEM. 2.12 Statistical analyses Data for viscosity, DC, cell viability, contact angle and microtensile bond testing were expressed as means ± standard deviations after ascertaining the normality and homoscedasticity of the data sets. One-way analysis of variance and Tukey’s multiple comparison tests were used to determine differences among groups using SPSS 19.0. Failure modes were analyzed using the chi-square method. All statistical analyses were performed at a confidence level of 95%. 3. Results 3.1 UMP Characterization Figure 2 shows the infrared spectrum of UMP with characteristic -NH stretching vibration (3356.2 cm-1) and the amide II (1524.3 cm-1) peaks. The appearance of these peaks confirmed the reaction between HEMA and IPDI. The peak at 2257.4 cm-1 suggested the existence of a -NCO

12

group in UMP. Absence of a peak at 3429.8cm-1 indicated that HEMA was completely consumed. The peak at 1710.2cm-1 was designated to the -C=O group of UMP. Proton NMR spectra of UMP is depicted in Fig. 3. In the UMP spectrum, absence of a peak at 2.55 ppm indicated the lack of an -OH proton, which is indicative of the absence of HEMA. The singlet signal at 3.05 ppm was attributed to the proton of CH2 adjacent to –NCO. The signals at 4.60-4.69 ppm were attributed to the proton of -NH adjoined to aliphatic cyclic, and the signals at 4.88-4.91 ppm were attributed to the proton of -NH adjoined to methylene. Emergence of these two peaks confirmed the existence of UMP. Similar results were obtained by 13CNMR (Supplementary date: Suppl.-Fig.2). Results of high-resolution mass spectrometry are presented in Fig. 4. The major peaks represented were mono-substituted IPDI (HRMS (ESI) m/z calculated for C18H28N2O5Na [M+Na]+ 375.1890, found 375.1886) and di-substituted IPDI (HRMS (ESI) m/z calculated for C24H38N2O8Na [M+Na]+ 505.2520, found 505.2539). 3.2 Viscosity and Degree of conversion The viscosity values of the tested resin monomers (expressed in means ± standard deviations) are presented in Table 1. The viscosity of the UMP was 1.25 Pa-s. Compared with the other tested methacrylate resin monomers, Bis-GMA had the highest viscosity, which was significantly different from that of UMP (P < 0.01). Both HEMA and TEGDMA had much lower viscosity than the other resin monomers (P < 0.05) and were not significantly from one another. The UMP had the highest DC value (87.9 ± 0.8) among all the resin monomers (P < 0.001) (Table 1). The DC decreased in the order: UMP > HEMA > TEGDMA > UDMA > BisGMA (P < 0.001).

13

Table 1. Properties and degree of conversion of UMP and common methacrylate resin monomers Monomer/precursor

Viscosity (Pa-s)

Degree of conversion (%)

Molecular weight (Da)

UMP

1.25 ± 0.01a

87.9 ± 0.8a

350-480

Bis-GMA

1206 ± 5b

34.8 ± 0.5e

510.6

UDMA

27.8 ± 0.3c

62.9 ± 0.5d

470

TEGDMA

0.02 ± 0.00d

73.3 ± 0.7c

286.3

HEMA

0.01 ± 0.00d

84.7 ± 0.8b

130.14

Different case letters in the same column indicate statistically significant differences (P < 0.05) 3.3 Cytotoxicity Viability of the hDPSCs exposed to various resin monomers after 24 h are shown in Fig. 5. Cell viability decreased in a dose-dependent manner for all the resin monomers examined. Cells exposed to UMP showed cell viabilities ranging from 106.6 to 62.5% at concentrations less than 120 μg/mL and from 51.3 to 44.8% at concentrations between 140 and 160 μg/mL. Thus, 140160 μg/mL represents the median lethal concentration (LC50) for UMP. The LC50 for Bis-GMA was between 40 and 60 μg/mL and for Bis-EMA was 120–140 μg/mL. UDMA and TEGDMA showed similar cytotoxicity as UMP, which had LC50 values at approximately 140 μg/mL. TEGDMA showed the highest cell viability at concentrations ranging from 80 to 180 μg/mL among all the resin monomers (P < 0.01). 3.4 Interaction between demineralized dentin and UMP The disappearance of the -NCO peak and the appearance of a urea -C=O peak (1652.6cm-1) confirmed the reaction between UMP and dry demineralized dentin (Fig. 6; Table 2). New urethane -C=O peak (1719.2cm-1) and ester -C=O peak (1734.6cm-1), which were originated from UMP, appeared in UMP treated dry demineralized dentin. The appearance of these new 14

peaks indicating that UMP was successful grafted onto dentin collagen. The amide I peak of the dry demineralized dentin (1632.5cm-1) shifted to a lower wavenumber (1627.3cm-1) after the reaction. Infrared spectra of the interaction between UMP and moist demineralized dentin also showed the disappearance of the -NCO peak and the appearance of the urea -C=O peak (1645.8cm-1), urethane -C=O peak (1700.0cm-1) and ester -C=O peak (1730.2cm-1). In contrast, the amide I peak shifted to a higher wavenumber (1634.0 cm-1). The ratio of CH2 scissoring /amide III was stable and showed no statistical difference (P > 0.05) before and after the interaction in each group. Table 2. Characteristic infrared absorbance peaks of demineralized dentin, UMP-demineralized dentin and ratio of ACH2 scissoring /Aamide III under different conditions Group

Urea C=O

Urethane Ester C=O Amide I C=O

Dry demineralized dentin

-

-

-

1632.5

1548.5

0.98 ± 0.02a

UMP-dry demineralized dentin

1652.6

1719.2

1734.6

1627.3

1543.2

0.97 ± 0.02a

Moist demineralized dentin

-

-

-

1633.3

1554.0

0.98 ± 0.01b

UMP-moist demineralized dentin

1645.8

1700.0

1730.2

1634.0

1539.5

0.97 ± 0.01b

Amide II

CH2 scissoring /Amide III

Same superscript letters above the columns indicate no significant difference (P > 0.05) 3.5 Degree of conversion of adhesive Table 3 reports the degree of conversion of the experimental and control adhesives. The degree of conversion of the experimental adhesive was slightly higher than of Single Bond 2 (P < 0.01). Table 3. The formula, degree of conversion, micro-tensile bond strength, and fracture mode of each tested adhesive. Group

EXP SB2

Composition UMP (40%), Bis-EMA (40%), acetone (20%), CQ (0.5%), EDMAB (0.5%) Bis-GMA, HEMA,

Degree of conversion (%)

Microtensile strength (MPa)

Fracture mode (A/ D/ C/ M) (%)

61.0 ± 0.3a

34 ± 4a

30/0/10/60a

59.9 ± 0.3b

27 ± 5b

35/5/10/50a

15

dimethacrylates, silica nanofiller, polyalkenoic acid copolymer, initiators, water, ethanol

EXP: experimental UMP-containing adhesive; SB2: Single Bond 2. Failure mode: (A) adhesive failure; (D) cohesive with dentin; (C) cohesive with resin composite; (M) mixed failure involving both interfacial and cohesive fracture. Different case letters in the same column indicate statistically significant differences (P < 0.05). 3.6 Contact angle As shown in Fig. 7, the contact angle of the experimental adhesive on demineralized dentin was significantly higher than of SB2 (P < 0.001). Likewise, the water contact angle of SB2treated dentin was much lower than that of the experimental adhesive-treated dentin (P < 0.001). These results are indicative of a markedly more hydrophobic interface in demineralized dentin bonded with the experimental adhesive. 3.7 Microtensile bond strength and failure mode Results derived from dentin bonded with the experimental and control adhesives are depicted in Table 3. The immediate tensile bond strength for the experimental adhesive was 34 ± 4 MPa, which was significantly higher than that of SB2 (27 ± 5 MPa; P < 0.05). The type of adhesive used for bonding dentin did not influence failure mode distribution (P > 0.05). Most of the specimens exhibited a mixed failure mode for both adhesive groups. 3.8 Morphologic examination Representative FE-SEM images of the resin-dentin interface created with the adhesive systems employed in the present study are shown in Fig. 8. Both SB2 and the experimental UMP-containing adhesive produced a uniform hybrid layer and abundant resin tags. Long cylindrical resin tags with side branches were identified in the experimental adhesive group (Fig.

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8 A, B, E, F). Single Bond 2-treated dentin showed funneled and rough resin tags with pronounced infiltration depth (Fig. 8 C, D, G, H). 4. Discussion Dental adhesives have contributed significantly to contemporary prosthetics and restorative dentistry. Different types of dentin adhesives are available based on the interaction between target tissues (enamel, dentin, oral mucosa) and exogenous materials (resin, ceramic, metal). Bio-adhesives used to bridge endogenous tissues with exogenous materials should maximize the application of as many potential bonding mechanisms as possible [34-37]. During dentin bonding with the etch-and-rinse technique, unprotected collagen fibrils inevitably remain in the bonded interface and are exposed to an easily-degradable micro-environment. Although self-etch adhesives containing some functionalized phosphate resin monomers can bond chemically to carbonated apatite, the reaction potential of those resin monomers with collagen is not fully utilized and the collagen matrix is not chemically united with the adhesive matrix. Resin monomers with the capacity to bridge dentin collagen with the adhesive resin matrix via copolymerization would theoretically produce more stable interfaces that are more resistant to degradation. In the present study, resin monomer precursors containing 20 wt% -NCO functional groups and polymerizable methacrylate groups were synthesized via nucleophilic addition reaction between IPDI and HEMA. In the reaction system, IPDI was used as the reagent and DBTBL was utilized as the catalyst. The choice of IPDI was based upon its previous utilization in the biomedical domain, such as drug releasing systems and cell scaffolds [38, 39]. The IPDI molecule contains both an aliphatic and a cycloaliphatic -NCO group, endowing it with the potential of reacting with the -OH and -NH2 side chains of amino acids present in the collagen 17

molecule. The aliphatic -NCO group is effectively shielded by the β-substituted methyl group, the cyclohexane ring, and the neighboring methyl group. In contrast, the cycloaliphatic -NCO is in the activated transition state and is much more reactive than the aliphatic -NCO [40]. When DBTBL was used as the catalyst, the ratio of the rate constant augmented to 11.5. The reaction kinetics of IPDI and HEMA may be explained by a second-order kinetic equation. During the first stage of the carbamate-yielding reaction, the nucleophilic center of the -OH in HEMA is added to the electrophilic carbon atom in the carbonyl group of IPDI and the proton is synchronously transferred to the nitrogen atom. In addition, -OH and -NHCOO have an autocatalytic effect. Both external catalysts and urethane groups influence the reaction kinetics [41]. The urethane was prepared in a 2/1 ratio of NCO/OH to ensure that some terminal isocyanate groups remained free. The free terminal isocyanate groups form covalent bonds with the -NH2 group of dentin collagen [28]. The -CONH and -NCO of UMP were confirmed by ATR-FTIR and proton NMR. Monomer viscosity is closely related to features such as the degree of conversion, flexibility and mechanical properties [42]. Because viscosity is dependent on molecular weight, fluids with higher molecular weight usually possess higher viscosity [43]. In addition, viscosity reflects the resistance of molecules to flow, and a fluid with high viscosity usually indicates the existence of intermolecular interactions. The synthesized UMP exhibited a higher viscosity than TEGDMA and HEMA, because of hydrogen bonding between -NH and -C=O. The hydrogen bonds in NHCOO may enhance adhesive strength and stability [44]. However, the viscosity of UMP is far less than that of Bis-GMA; the very high viscosity of Bis-GMA is attributed to its hydroxyl groups and the two core aromatic rings [45, 46]. The relative lower viscosity of UMP may also be attributed to the flexibility of the urethane groups [47, 48]. Monomers conversion into stable,

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high-molecular weight polymers constitutes the tenet of stable dental adhesion [49]. Compared with other methacrylate resin monomers, UMP had the highest DC. This may be explained by UMP’s relatively high molecular flexibility and the increased chance of molecule collision. Unreacted resin monomers in resin-based dental materials may leach into the pulp via dentinal tubules. Leached resin monomers adversely affect vital pulp tissues especially during the first 24 h [50, 51]. All the tested resin monomers showed reduction in cell viability in a concentration-dependent manner, with different concentration ranges. The LC50 was in the order of Bis-GMA (40–60 μg/mL) < UDMA, Bis-EMA (120–140 μg/mL) < UMP (140–160 μg/mL) < TEGDMA (higher than 180 μg/mL). The present results are in line with those reported in other studies [52-55]. Compared with UDMA, Bis-GMA causes deeper cell penetration because of its higher lipo-solubility [54]. Based on the LC50 data, the cytotoxicity of UMP should be comparable with UDMA because of their similar structure. Considering that UMP is more viscous and has a higher degree of conversion that TEGDMA, UMP is less likely to be released into the pulp from the resin matrix; this hypothesis should be further tested for confirmation. Urethane-based modification has been used to decrease the toxic effect caused by commercially available Bis-GMA and TEGDMA [56]. Polymerized urethane-modified Bis-GMA-based resins showed 50% less leaching of unreacted resin monomers during extraction by organic solvents, are potentially less toxic than Bis-GMA when used in close vicinity with vital dental pulps [56]. The major organic component of dentin is type I collagen [21]. The basic unit of collagen is (Gly-Xaa-Yaa)n, where X and Y are predominantly proline (Pro) and hydroxyproline (Hyp) [57]. These amide acid residues provide ample -NH2 groups for covalent bonding with the -NCO group of UMP. The ATR-FTIR results indicate that two types of reaction may occur between UMP and completely demineralized dentin. Disappearance of -NCO and appearance of the urea 19

C=O peak after the reaction indicate that there was covalent bonding between the -NCO and the NH2 groups of dentin collagen. The appearance of new urethane -C=O and ester -C=O peaks in UMP-treated dry and moist demineralized dentin, originated from UMP, is indicative of successful grafting of UMP onto dentin collagen. As one of primary interactions, the covalent bond is a high energy bond and usually produces stronger adhesion [58]. Apart from covalent bonding, the amide I peak shifted toward a low wavenumber following the interaction of UMP with dry demineralized collagen. This may be attributed to new hydrogen bond formation between the UMP and collagen. The donated proton is derived from the hydrogen atom of the NH group and the proton acceptor may be the carbonyl (-C=O) group. Although the energy of the hydrogen bond is much lower than that of the covalent bond, it is significant for bonding when a large number of sites are available for hydrogen bonding within the resin-dentin interface [58]. Recent studies suggest that hydrogen bonding is the main cross-link mechanism between the hydroxyl groups present in plant-derived cross-linking agents (e.g. proanthocyanidin) and the amino and amide groups of collagen [59]. The CH2 scissoring/amide III ratio did not change significantly after the interaction of UMP with collagen, which is indicative of a relative stable triple helix structure of collagen. According to the aforementioned results, the UMP may be cross-linked to collagen via covalent bond and hydrogen bond formation. This reaction mechanism is similar to hexamethylene diisocyanate, another -NCO based polymer that has been used extensively for cross-linking collagen-based biomaterials [28, 60]. A polyurethane-based root canal obturation material, which has the same urethane (-NHCOO-) functionality as UMP, was found to have excellent push-out bond strengths with root dentin [61]. This may be attributed to the stronger bond formed between the polyurethane-based material and the root dentin collagen matrix [61].

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An experimental adhesive was formulated to examine the potential bonding efficacy of UMPcontaining adhesive to acid-etched dentin. Because there is no commercial Bis-EMA-based etchand-rinse adhesive available to date, it is not possible to select a Bis-EMA-based commercial adhesive as the control group. Single Bond 2, a popular two-step etch-and-rinse adhesive, was used as the alternative control to evaluate whether the experimental adhesive was feasible for clinical use. Our results showed that the experimental adhesive containing 40% UMP produced higher microtensile bond strength to dentin compared with the commercially available control adhesive. This promising result may be attributable to several factors. The experimental adhesive has a higher degree of conversion, which may play a crucial role in interfacial strength [62]. This may be explained by the greater molecular mobility caused by the lower viscosity of Bis-EMA and UMP in the experiment adhesive, compared with SB2 which contains less flexible Bis-GMA [63]. Acetone has a very good water-chasing effect, due to its high dipole moment and high vapor pressure, which is approximately four times as high as that of ethanol [16]. Formation of water-acetone azeotrope results in water loss during acetone evaporation. Hence, remnant water that resides within the bonding interface after acid-etching and rinsing may be removed more effectively. Less residual solvents within the interface may lead to a higher degree of conversion of the adhesive, especially at the base of the hybrid layer [64]. The ATR-FTIR results also demonstrated reduction in hydrogen bond content when moisture-demineralized dentin was employed as the bonding substrate, partially confirming the reduction in moisture. Previous studies reported that acetone is not capable to re-expanding shrunken demineralized collagen due to its low hydrogen bonding capacity, leading to poor infiltration of the adhesive monomers [65]. This adverse effect may be offset by cross-linking collagen with UMP, producing a collagen matrix with increased hydrogen bonding capacity. This speculation requires further verification.

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Complete infiltration of adhesive resin monomers into the demineralized dentin collagen network is essential for creating strong adhesion and for enveloping individual collagen fibrils [21]. The low contact angle of the experimental adhesive on demineralized dentin indicates its capacity to infiltrate the demineralized dentin substrate. These results are also consistent with the FE-SEM observations, in which long, homogeneous cone-shaped resin tags were present for effective micromechanical interlocking. The presence of these resin tags was indicative of good flowability of the preliminary UMP-based adhesive. With additional contribution derived from chemical bonding between the collagen substrates and the adhesive, a reliable bonding interface may be produced. This was confirmed by the FE-SEM results, in which no obvious unexposed collagen fibrils could be identified when the experimental adhesive was applied. Although resin infiltration was not hindered for this preliminary UMP-based adhesive, the reaction kinetics for UMP should be further studied in the case that too rapid on-site reaction may clog the tubules, which may adversely affect the depth of resin penetration. It is intriguing to find that the polymerized experimental adhesive on demineralized dentin surface was much more hydrophobic than polymerized SB2. This may be partly explained by the incorporation of a hydrophilic polyalkenoic acid copolymer in SB2, which enhances the wetting ability of the adhesive [66]. A less hydrophilic interface reduces water sorption, which is paramount in increasing interfacial bond stability. In addition, studies have shown that collagen cross-linking enhances the stiffness of collagen, rendering it less susceptible to enzymatic degradation. Urethane-based materials possess high abrasion resistance, toughness, chemical resistance and mechanical strength [61, 67-69]. A new urethane-based monomer, 1,1,1-tri-[4(methacryloxyethylamino-carbonyloxy)-phenyl] ethane that was synthesized for incorporation into dental adhesives demonstrated improved esterase resistance [69]. Thus, the UMP in the

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present study may have the advantage of maintaining the long-term stability of the resin-dentin interface. Investigations regarding the durability of resin-dentin interfaces created by UMP-based adhesives are in order. 5. Conclusions In the present study, UMP with 20 wt% isocyanate group and a methacrylate group were synthesized. The isocyanate-terminated urethane with functionalized methacrylate groups demonstrated viscosity, degree of conversion and biocompatibility that are comparable with commercially available methacrylate resin monomers. Methacrylate-functionalized UMP reacts with dental collagen via covalent and hydrogen bonds. An experimental adhesive containing 40% UMP demonstrated co-polymerization capability, good infiltration capacity and achieved higher immediate bond strength to dentin than the control commercially available adhesive. Improvement of dentin bonding by incorporation of UMP into dentin adhesives justifies future evaluation of the potential of these UMP-based adhesives in extending the longevity of resindentin bonds. Within the limitations of the present work, the use of UMP promotes dentin bonding and warrants further studies with respect to whether the more hydrophobic resin-dentin interface created with UMP-containing adhesives are less susceptible to water sorption and more resistant to degradation by host-derived endogenous proteases. Acknowledgments This work was supported by grants from the National Key R&D Program of China (2017YFC0840100 and 2017YFC0840109), National Natural Science Foundation of China (81720108011 and 81470773) and the program for Changjiang Scholars and Innovative Research Team in University (No. IRT13051).

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Conflicts of Interest The authors declare no competing interest.

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Figure legends Fig. 1. Synthesis of isocyanate-terminated urethane methacrylate precursors. Fig. 2. Infrared absorbance spectra of HEMA, IPDI and UMP. Fig. 3. 1HNMR spectra of UMP. Fig. 4. HRMS spectra of UMP. (A) mono-substituted IPDI (C18H28N2O5Na [M+Na]+). (B) disubstituted IPDI (C24H38N2O8Na [M+Na]+). Arrows indicate the peaks of the two components. Fig. 5. Cell viability of hDPSCs exposed to (A) UMP, (B) Bis-GMA, (C) UDMA, (D) TEGDMA and (E) Bis-EMA at various resin monomer concentrations. The red dotted line represents 50% cell viability. Fig. 6. ATR-FTIR of UMP-dentin interaction (A) UMP reacted with dry demineralized dentin. (B) UMP reacted with moist demineralized dentin. Fig. 7. (A) Contact angle of adhesives on demineralized dentin. (B) Contact angles of water on dentin treated with adhesives. DD: demineralized dentin; EXP: experimental UMP-containing adhesive; SB2: Single Bond 2. Fig. 8. Representative FE-SEM images of the resin-dentin interface. (A), (B) experimental adhesive-treated dentin and (C), (D) Single Bond 2-treated dentin. (E), (F), (G), and (H) are high magnification images of (A), (B), (C), and (D), respectively. Supplementary Fig. 1. 1HNMR spectra of (A) HEMA and (B) IPDI. The singlet signal at 2.55 ppm was attributed to the terminal -OH in HEMA. Multiplet signals such as the peaks at 3.65 ppm and 3.53 ppm suggest that the proton on the aliphatic cycle adjacent to -NCO represents two signals on the 1HNMR, indicating the existence of IPDI isomer.

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Supplementary Fig. 2.

13CNMR

of (A) HEMA, (B) IPDI and (C) UMP. The

13CNMR

was

examined with a spectrometer (ASCENDTM 600, Bruker, Rheinstetten, Germany) and deuterated CDCl3 was used as solvent. From the 13CNMR of UMP, the H+H’ were attributed to the carbon of -NCO and the carbon of methylene adjacent to aliphatic cycle. The F+F’ were assigned as the carbon of -CO-NH. These peaks are indicative of the existence of monosubstituted IPDI and di-substituted IPDI. Supplementary Fig. 3. High resolution mass spectroscopy of (A) IPDI, (B) HEMA and (C) UMP. The peaks are indicative of the presence of IPDI (HRMS (ESI) m/z calculated for C12H18N2O2H+ [M+H]+ 223.1441, found 223.1450) and HEMA (HRMS (ESI) m/z calculated for C6H10O3H+ [M+H]+ 131.07027, found 131.07082). The spectra of UMP did not show any characteristic peaks of IPDI and HEMA, suggesting complete reaction between HEMA and IPDI. Composite-adhesive restorations have become an indispensable treatment modality in contemporary restorative dentistry. While the inability of these adhesives to bond chemically with collagen undermines the bond quality. This study describes a novel isocyanate-terminated urethane-multi-methacrylate precursors (UMP) which can bridge dentinal matrix collagen with adhesive resin by covalent and hydrogen bonds. Furthermore, an experimental UMPbased adhesive shows better co-polymerization capability, good infiltration capacity and higher immediate bond strength than the putatively effective adhesive Single Bond 2. The new chemical bonding mechanism based on UMP would theoretically produce more stable bonding interface that are more resistant to degradation.

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