Synthesis and characterization of triethylene glycol dimethacrylate nanocapsules used in a self-healing bonding resin

Synthesis and characterization of triethylene glycol dimethacrylate nanocapsules used in a self-healing bonding resin

Journal of Dentistry, 2011-12-01, Volume 39, Issue 12, Pages 825-833, Copyright © 2011 Elsevier Ltd

Abstract

Objectives

To date, the production of highly durable dentine bonding is still a challenge. Self-healing bonding resins may provide a new direction for the improvement of the bonding durability. The objective of the current study was to synthesize polyurethane nanocapsules encapsulated with the core material triethylene glycol dimethacrylate (TEGDMA) for use as a major component in a self-healing bonding resin.

Methods

TEGDMA nanocapsules were synthesized via interfacial polycondensation in a miniemulsion, and the TEGDMA nanocapsules were then characterized via Fourier-transform infrared (FTIR) spectrometer, field emission scanning electron microscopy (FESEM), and high-performance liquid chromatography (HPLC) to investigate the morphology, the average TEGDMA loading ( DL %), and encapsulation efficiency ( EE %). The mechanical property of dental adhesive with different concentrations (0, 3, 6, 9, and 12 wt%) of the TEGDMA nanocapsules were also measured, and the cytotoxicity was investigated using an MTT assay.

Results

FTIR confirmed that the TEGDMA nanocapsules were successfully synthesized. These nanocapsules showed a high drug load. The bond strength of the dental adhesive incorporated with 9 wt% TEGDMA nanocapsules was significantly higher compared with those of the other groups ( P < 0.001). Moreover, the biocompatibility of the dental adhesive was not affected by the incorporation of the TEGDMA nanocapsules.

Conclusions

The current study demonstrated the successful synthesis of TEGDMA nanocapsules, and the overall properties of the dental adhesive were not compromised.

Introduction

The immediate bonding effectiveness of contemporary dentine adhesives has become favourable with the development in adhesive dentistry. However, the limited durability of dentine adhesives in vivo is still a major shortcoming. A number of factors affect the durability of adhesives in clinical applications, such as the degradation of exposed collagen fibrils, especially for etch and rinse adhesives, and the hydrolytic breakdown of adhesive monomers primarily for self-etch adhesives. Microcracking induced by thermal and mechanical fatigue is also a long-standing problem that affects the durability of adhesives. At present, methods of enhancing the durability primarily focus on the built-in dentine-associated matrix metalloproteinases (MMPs) inhibitors or the improvement of the bonding technique, such as ethanol wet-bonding. However, there are little studies focused on enhancing the durability by healing the microcracks.

Tensile loading, fatigue loading, and thermal cycling can result in the formation of microcracks in dental adhesives. The nucleation of microcracks around weak links can propagate continuously, resulting in the degradation of mechanical properties, formation of pathways for the entry of liquids and bacteria, and complete material failure. Studies have indicated that the weak links in dental bonding adhesion are located in the hybrid layer, the quality of which is important to bond strength. Therefore, to improve the durability of dental adhesives, microcracks should be repaired before the integrity of the adhesive is destroyed.

Scientists have recently proposed self-repair ideas that may increase the life expectancy of composite materials. The discovery of a self-healing resin based on the use of microcapsules is a research breakthrough. White et al. successfully synthesized a self-healing epoxy resin composite through the encapsulation of a healing agent within the microcapsules. These encapsulations were embedded with a catalyst capable of polymerizing the healing agent in a polymer matrix. The rationale for the self-healing design is that when cracks form in the matrix, they rupture the microcapsules, releasing the healing agent into the crack plane through capillary action. The healing agent then comes into contact with the catalyst, triggering polymerization, and finally bonds until the crack faces close.

To date, the use of a self-repair bonding resin to improve the durability of dental adhesives is an open and intriguing approach. Meanwhile, the rapid development in nanoscale controlled drug delivery technology provides the possibility of a ‘smart’ material for the purposes of self-healing in the oral environment.

Scientists have postulated that a chemical repair monomer encapsulated in a polymer can be an important factor in developing self-repair materials. Polyurethanes, an important class of polymers, are widely used in the medical field to encapsulate a wide range of active agents. The excellent physicomechanical properties, high flexibility, and relatively good biocompatibility of polyurethanes make them suitable shell materials for encapsulation and meet the requirements of self-healing nanocapsules. Moreover, triethylene glycol dimethacrylate (TEGDMA) is a frequently used monomer with low viscosity, making it a suitable candidate as a core material. Therefore, a hypothesis on the synthesis of nanocapsules encapsulated with an active monomer that exhibits the self-healing function of dental adhesives based on the aforementioned rationale is proposed.

The primary objective of this pilot study was to investigate the synthesis and characterization of polyurethane nanocapsules containing TEGDMA via interfacial polycondensation in a miniemulsion for use as a major component in self-healing bonding resins. The mechanical properties and cytotoxicity were also determined to investigate the compatibility of TEGDMA nanocapsules with a commercial dental adhesive.

Materials and methods

Materials

Isophorone diisocyanate (IPDI) and TEGDMA were obtained from Aldrich Chemical Company, Inc., USA. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma Chemical Company. Dulbecco’s modified Eagle’s medium (DMEM) and foetal bovine serum (FBS) were from Gibco Company. All other reagents were of analytical grade.

Fabrication of the TEGDMA nanocapsules

The TEGDMA nanocapsules were encapsulated in polyurethane by interfacial polycondensation in miniemulsion of IPDI and 1,6-hexanediol as reported by Torini et al. Detailed process could be seen in our precious work and the brief procedure was showed in Fig. 1 .

Scheme of synthesis of TEGDMA-nanocapsule 0.88 g of SDS and 0.66 g HD were dissolved in 70 ml distilled water, and stirred by magnetic stirring apparatus at 300 rpm, 40 °C for 1 h. And then 6.66 g of TEGDMA and 2.22 g of IPDI were added and stirred for 10 min under the same conditions, after which high speed shear and ultrasonication were proceeded to form miniemulsion. At the end of ultrasonication, 5.91 g of 1,6-hexanediol solvent was slowly added to the miniemulsion. Through demulsification, centrifugation, rinse and lyophilisation, the TEGDMA nanocapsules were fabricated.
Fig. 1
Scheme of synthesis of TEGDMA-nanocapsule 0.88 g of SDS and 0.66 g HD were dissolved in 70 ml distilled water, and stirred by magnetic stirring apparatus at 300 rpm, 40 °C for 1 h. And then 6.66 g of TEGDMA and 2.22 g of IPDI were added and stirred for 10 min under the same conditions, after which high speed shear and ultrasonication were proceeded to form miniemulsion. At the end of ultrasonication, 5.91 g of 1,6-hexanediol solvent was slowly added to the miniemulsion. Through demulsification, centrifugation, rinse and lyophilisation, the TEGDMA nanocapsules were fabricated.

Morphological analysis

The TEGDMA nanocapsules were dispersed in distilled water as mentioned above and then one droplet was dropped on tin foil. The sample was dried at room temperature and observed under FESEM (Sirion 200, FEI, Netherlands) at 15 kV.

TEGDMA loading and encapsulation efficiency

Nanocapsules loaded with TEGDMA (10 mg) were dissolved in isopropanol followed by 30 min on ultrasounds. After extraction, it was diluted to volume with distilled water to 10 ml. Then 1 ml solution was extract and diluted to volume with mobile phase to 10 ml. The resulting solution was filtered through 0.45 μm PTFE membrane filters. And then the filtered solution (20 μl) was injected into a high performance liquid chromatographic (HPLC) apparatus. And the amount of TEGDMA entrapped was determined by HPLC. A ChromoQuest thermoquest France SA instrument was used with Agilent HC C-18 (5 μm, 250 mm × 4.6 mm). The mobile phase consisted of methyl cyanides and water (55:45). The flow rate was 1 ml/min. A volume of 20 μl loop was injected at temperature of 25 °C. TEGDMA was detected using UV spectrophotometer (Secomam, Anthelie, France) at the λ max value of 205 nm and results were quantified through the use of the equation derived from the slope of the standard curve prepared for TEGDMA ( R 2 = 0.9998) at 205 nm. These data were collected in triplicate, but the average values were considered in calculating % drug loading and encapsulation efficiency. These were measured using the following equations:

DL%=WLWT×100% D L % = W L W T × 100 %
DL is the TEGDMA loading; W L is the weight of TEGDMA in TEGDMA nanocapsules; and W T is the weight of TEGDMA nanocapsule powders.
EE%=WLWa×100% E E % = W L W a × 100 %
EE is the encapsulation efficiency; W a is the initial weight of TEGDMA taken for loading; and W L is the weight of TEGDMA in TEGDMA nanocapsules.

Polymer characterization

Fourier transform infrared ray (FTIR) spectroscopy (Vertex 70, Bruker, Ettlingen, Germany) was performed using a potassium bromide disc on a spectrometer with a resolution of 4.00 cm −1 . Infrared spectra were recorded in the range of 500–4000 cm −1 to evaluate the molecular structure and phase clarification of blank nanocapsules and TEGDMA nanocapsules, respectively.

Micro-tensile bond testing

Twenty freshly extracted human third molars were used in this test. The teeth were collected after the patients’ informed consent was obtained under a protocol approved by the Ethics Committee for Human Studies, School of Stomatology, Wuhan University. The occlusal one-third of the crown was removed by a water-cooled low-speed diamond saw (Isomet, Buehler, Evanston, IL, USA). A smear layer was created by abrading the dentine with 600 grit SiC paper under water. The prepared dentine specimens were randomly divided into five groups. Bonding procedures were performed on the treated occlusal surfaces of mid-coronal dentine with Prime & Bond NT (Dentsply De Trey, Germany) (NT) in the absence (control, Group A) or the presence of 3% (Group B), 6% (Group C), 9% (Group D) and 12% (Group E) of the TEGDMA nanocapsules. Composite buildups were performed using Charisma universal light curing Microglass ® composite (Heraeus Kulzer, Germany). For μTBS evaluation, composite-dentine beams of 0.8 mm 2 were stressed to failure at a crosshead speed of 1 mm/min ( n = 20 per group). The μTBS data were analysed by one-way ANOVA and Tukey’s multiple comparison tests to determine any significance at 5% level.

For the adhesive/dentine interfaces ultrastructure observation, intact specimens of each group were kept in advance. The adhesive/dentine interfaces and the surfaces of fractured specimens were sputter-coated with gold/palladium and examined with FESEM at 15 kV, respectively.

MTT assay

Cytotoxicity of TEGDMA nanocapsules was identified measuring cell survival and proliferation in vitro by MTT assay, as previously reported. Mouse 3T3 fibroblasts were placed in a 96-well tissue culture dish at 1 × 10 4 cells/well. After 24 h of incubation, the medium was removed and cell monolayer was incubated in the absence of the control (Group 1) or the presence of extracts from Prime & Bond NT (Group 2), Prime & Bond NT + TEGDMA nanpcapsules (Group 3), and Prime & Bond NT + blank nanocapsules (Group 4) for 1 d, 3 d, and 5 d, respectively. The medium was replaced by 20 μL/well of a solution of MTT (0.5 mg/ml) in phosphate buffered saline (PBS), and the cells were incubated at 37 °C for 4 h in a 5% CO 2 atmosphere. The MTT solution was removed and replaced with 150 μL/well of DMSO and gently shaken until the crystals were dissolved. The optical density (OD) was immediately measured in a Bio-Rad ELISA reader at 490 nm. Each experiment was performed in triplicate.

Results

Morphology of TEGDMA nanocapsules

The FESEM micrographs exhibit that the TEGDMA nanocapsules have a well-developed spherical shape and a smooth surface ( Fig. 2 A and B). No obvious coalescence phenomena and fragments are observed.

FESEM micrographs of nanocapsules (A and B): FESEM micrographs indicate that the TEGDMA nanocapsules are spherical with smooth and condense surface.
Fig. 2
FESEM micrographs of nanocapsules (A and B): FESEM micrographs indicate that the TEGDMA nanocapsules are spherical with smooth and condense surface.

TEGDMA loading and encapsulation efficiency

The mean TEGDMA loading ( DL %) and the average encapsulation efficiency ( EE %) are (47.7 ± 2.2) % and (90.5 ± 2.2) %, respectively.

Structure of TEGDMA nanocapsules

FTIR spectrums of the blank nanocapsules and TEGDMA nanocapsules are exhibited in Fig. 3 . In Fig. 3 A, the spectrum shows absorption bands for the N–H vibration at 3370 cm −1 , the C O vibration of urethane at 1702 cm −1 , the urea carbonyl vibration at 1640 cm −1 , and the C–N vibration at 1561 cm −1 . The TEGDMA nanocapsule presents characteristic absorption peaks of TEGDMA at 1637 cm −1 (overlapped with the urea carbonyl absorption band at 1640 cm −1 ) for the C C group and at 1171 cm −1 for the C–O–C group ( Fig. 3 B).

FTIR spectrums of nanocapsules: A – blank nanocapsules, B – TEGDMA nanocapsules. (A) The spectrum shows absorption bands for the N–H vibration at 3370 cm −1 , the C O vibration of urethane at 1702 cm −1 , the urea carbonyl vibration at 1640 cm −1 , and the C–N vibration at 1561 cm −1 . (B) Strong peaks at 1171 cm −1 associated with TEGDMA can be observed.
Fig. 3
FTIR spectrums of nanocapsules: A – blank nanocapsules, B – TEGDMA nanocapsules. (A) The spectrum shows absorption bands for the N–H vibration at 3370 cm −1 , the C O vibration of urethane at 1702 cm −1 , the urea carbonyl vibration at 1640 cm −1 , and the C–N vibration at 1561 cm −1 . (B) Strong peaks at 1171 cm −1 associated with TEGDMA can be observed.

Micro-tensile bond strength (μTBS) test

Mean μTBS values obtained in all experimental groups are displayed in Fig. 4 . For Group A, the μTBS is 20.3 ± 4.9 MPa. For Group B, Group C, and Group E, the bond strengths are 19.2 ± 5.3 MPa, 17.8 ± 3.8 MPa, and 18.2 ± 3.2 MPa, respectively. The highest μTBS (25.8 ± 4.9 MPa) value is obtained from the adhesive contained 9 wt% nanocapsules ( P < 0.001). Typical fracture surfaces and adhesive/dentine interfaces are shown in Fig. 5 .

TBS values of five groups The μTBS of adhesive of Prime & Bond NT in the absence (control, Group A) or the presence of 3% (Group B), 6% (Group C), 9% (Group D) and 12% (Group E) of the TEGDMA nanocapsules (* P < 0.001, n = 20).
Fig. 4
TBS values of five groups The μTBS of adhesive of Prime & Bond NT in the absence (control, Group A) or the presence of 3% (Group B), 6% (Group C), 9% (Group D) and 12% (Group E) of the TEGDMA nanocapsules (* P < 0.001, n = 20).
FESEM observations of the adhesive/dentine interfaces. (A and B) FESEM images of dentine (D) surface show that TEGDMA nanocapsule (black arrow) surrounded by adhesive are infiltrated into dentinal tubule (T) with resin tag (RT). (C and D) FESEM micrograph of the adhesive/dentine interface bonded with Prime & Bond NT or Prime & Bond NT incorporated with TEGDMA nanocapsules. The resin tags (white arrow) were longer and more regular as compared with the control. (E and F) Local magnification from (D) shows that TEGDMA nanocapsules (white arrows) could be notified on the surface of resin tag (RT). The rough and uneven surface indicates coverage of adhesive.
Fig. 5
FESEM observations of the adhesive/dentine interfaces. (A and B) FESEM images of dentine (D) surface show that TEGDMA nanocapsule (black arrow) surrounded by adhesive are infiltrated into dentinal tubule (T) with resin tag (RT). (C and D) FESEM micrograph of the adhesive/dentine interface bonded with Prime & Bond NT or Prime & Bond NT incorporated with TEGDMA nanocapsules. The resin tags (white arrow) were longer and more regular as compared with the control. (E and F) Local magnification from (D) shows that TEGDMA nanocapsules (white arrows) could be notified on the surface of resin tag (RT). The rough and uneven surface indicates coverage of adhesive.

Cytotoxicity test

The cytotoxicity data in this experiment are exhibited in Fig. 6 . The results demonstrate that extracts from NT with/without nanocapsules are cytotoxic with respect to control in the first 3 days under this experimental condition ( P < 0.05). However, eluates from Groups 1, 2, 3 and 4 are not significantly different from each other after 5 days ( P > 0.05). In Fig. 7 , it is observed that the cell number in Groups 2, 3, and 4 decreased compared with Group 1 during the first 3 days. When cultured for 5 d, there was no difference amongst the four groups.

OD values in MTT assay The proliferation of mouse 3T3 fibroblasts in the absence (Group 1) or the presence of extracts from Prime & Bond NT (Group 2), Prime & Bond NT + TEGDMA nanpcapsules (Group 3), and Prime & Bond NT + blank nanpcapsules (Group 4) for 1 d, 3 d, and 5 d, respectively (* P < 0.05).
Fig. 6
OD values in MTT assay The proliferation of mouse 3T3 fibroblasts in the absence (Group 1) or the presence of extracts from Prime & Bond NT (Group 2), Prime & Bond NT + TEGDMA nanpcapsules (Group 3), and Prime & Bond NT + blank nanpcapsules (Group 4) for 1 d, 3 d, and 5 d, respectively (* P < 0.05).
Representative images of 3T3 fibroblasts cultured in the absence (Group 1) or the presence of extracts from Prime & Bond NT (Group 2), Prime & Bond NT + TEGDMA nanocapsules (Group 3), and Prime & Bond NT + blank nanocapsules (Group 4) for 1 d, 3 d and 5 d, respectively.
Fig. 7
Representative images of 3T3 fibroblasts cultured in the absence (Group 1) or the presence of extracts from Prime & Bond NT (Group 2), Prime & Bond NT + TEGDMA nanocapsules (Group 3), and Prime & Bond NT + blank nanocapsules (Group 4) for 1 d, 3 d and 5 d, respectively.

Discussion

Providing simpler and faster-acting dental adhesives is necessary in the development of dental adhesives. However, optimizing the speed and efficiency should not jeopardize the quality and durability of dentine resin bonds. Considerable effort has been exerted to improve dentine bonding durability and stability. However, self-healing materials using nanocapsule technology have not yet been reported in the field of dental material research. In the current study, TEGDMA nanocapsules, which can be used as components for a self-healing bonding resin and may improve the durability of dentine bonding in the future, were successfully fabricated through interfacial polycondensation in miniemulsions.

Fabrication approaches for nanocapsules are well-documented, including interfacial polycondensation, interfacial polymerization, interfacial precipitation, interfacial deposition, and self-assembly procedures. In view of the liquid core material used in the present study, we synthesized TEGDMA nanocapsules via interfacial polycondensation in miniemulsions. Miniemulsions are dispersions of relatively stable oil droplets in water prepared by shearing a system containing oil, water, surfactant and costabilizer. Appropriate preparation and formation of miniemulsions suppress the coalescence of droplets and Ostwald ripening, resulting in the formation of latex particles and initial droplets with similar sizes as well as in a relatively narrow size distribution. Results from a previous study on nanocapsule size and PDI are consistent with this theory. In Fig. 2 , TEGDMA nanocapsules are shown to have a smooth surface and a well-developed spherical shape, both of which can facilitate the dispersion of nanocapsules into the dental adhesive. The dense shell of a TEGDMA nanocapsule shown in Fig. 2 B can protect the activity of TEGDMA.

Several studies have reported that high encapsulation efficiency nanoparticles were synthesized via miniemulsion polymerization. Both the drug loading and the encapsulation efficiency of the nanoparticles prepared via miniemulsion were higher than those obtained via emulsion. In the current study, the mean TEGDMA DL % and average EE % were (47.7 ± 2.2)% and (90.5 ± 2.2)%, respectively, which confirms the efficient encapsulation of TEGDMA in the nanocapsules. The relatively high TEGDMA DL % of the nanocapsules is important for the self-healing of dental adhesives.

The FTIR results show that polyurethane nanocapsules containing TEGDMA were successfully synthesized. The primary principle of polyurethane formation through interfacial polycondensation is that isocyanates react with the hydroxyl groups of diols and amino-functional molecules via addition to the carbon–nitrogen double bond. Theoretically, this reaction should not trigger the polymerisation of TEGDMA, which is usually polymerized via free radical polymerization, during synthesis. In Fig. 3 A and B, characteristic vibrations (3370, 1702, 1640, and 1561 cm −1 ) associated with the polyurethane structure confirm the formation of polyurethane nanocapsules. In Fig. 3 B, although the characteristic absorption peaks of TEGDMA at 1637 cm −1 corresponding to the C C group overlapped with the urea carbonyl absorption band at 1640 cm −1 , a strong peak at 1171 cm −1 associated with TEGDMA was observed, which confirms that TEGDMA was successfully encapsulated in the nanocapsules.

A dental adhesive or bonding resin is an intermediate resin layer between the composite resin and dentine. The primary role of this adhesive layer is to withstand the stress caused by the shrinkage of the composite during setting and to keep the restoration in place. In the current study, the microtensile bond test was selected to evaluate the performance of the dentine adhesive incorporated with TEGDMA nanocapsules. The microtensile bond strength of NT with 9 wt% TEGDMA nanocapsules was significantly higher (25.8 ± 4.9 MPa) compared with those of other groups ( P < 0.001), whereas the experimental adhesives containing 3, 6, and 12 wt% TEGDMA nanocapsules showed bond strengths similar to that of the control ( P > 0.05). The addition of particle fillers can result in an increase in fracture toughness via multiple phenomena, including crack pinning, crack bridging, microcracking, and crack deflection. The polyurethane shell material has a high level of flexibility and hardness; as the dimensions of the polyurethane capsules decrease, the load-bearing stress on the adhesive is reduced, thereby inhibiting crack formation and propagation. The spherical shape of the nanocapsules ( Fig. 2 ) provides smooth and rounded edges, resulting in an even distribution of stress throughout the adhesive.

Additionally, a number of studies reported that the microtensile bond strength is significantly correlated with the ultimate microtensile strength of the adhesive resin. The bond strength is possibly correlated with strength-based mechanical properties, such as flexural strength and fracture toughness. Thus, the polyurethane nanocapsule concentration possibly affects the toughness of the adhesives. However, the redundancy of the nanocapsules may cause aggregation and consequently compromise the mechanical property. Therefore, a 9 wt% nanocapsule content may be the optimum concentration. Future studies should focus on the detailed evaluation of the effect of these new nanocapsules on the material properties of adhesives with different volume fractions. Changes in the adhesive properties under ageing conditions should also be investigated.

FESEM images of the adhesive/dentine interfaces show different resin tag infiltrations ( Fig. 5 ). A number of studies found that a deeper penetration of the monomer into the demineralized dentine results in stronger micromechanical bonding. Evidence also indicates that bond strength relies on the quality of the hybrid layer and not on monomer infiltration. TEGDMA nanocapsules possibly aid monomer penetration without disturbing the hybrid layer. However, this finding remains to be verified.

Monomers can be released from dental resin materials and thus result in cytotoxicity in vitro and show certain adverse effects in vivo. Previous studies also demonstrated that modern dentine adhesives, including Prime & Bond NT, exhibit cellular toxicity. In the current study, the result of the MTT assay was consistent with those of previous studies. However, the addition of TEGDMA nanocapsules to this commercial adhesive did not affect cellular toxicity, indicating that TEGDMA nanocapsules met the biocompatibility requirements in clinical practice.

Conclusions

The synthesis and characterization of TEGDMA nanocapsules for use in a self-healing bonding resin were described in the current study. These nanocapsules showed not only a relatively narrow size distribution, high drug load, and good compatibility, but also potential mechanical enhancement of the bonding materials.

The present study is a preliminary research on the application of a self-healing bonding resin in an oral environment. Further studies have to be conducted to achieve the self-healing of dental materials. For example, investigating an effective initiator system for TEGDMA and the rupture of nanocapsules are issues that need to be resolved.

Acknowledgements

This work was supported by National Nature Science Foundation of China (No. 30672346 ) and Open Research Fund Program of Hubei-MOST & KLOBME under Grant No. 200903 . The authors thanked Dr. Yufeng Zhang and Prof. Tao Jiang for their valuable suggestions.

d Authors contributed equally to this work.

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Synthesis and characterization of triethylene glycol dimethacrylate nanocapsules used in a self-healing bonding resin Xiaobai Ouyang , Xueqing Huang , Qiuhua Pan , Chenqi Zuo , Cui Huang , Xiangliang Yang and Yanbing Zhao Journal of Dentistry, 2011-12-01, Volume 39, Issue 12, Pages 825-833, Copyright © 2011 Elsevier Ltd Abstract Objectives To date, the production of highly durable dentine bonding is still a challenge. Self-healing bonding resins may provide a new direction for the improvement of the bonding durability. The objective of the current study was to synthesize polyurethane nanocapsules encapsulated with the core material triethylene glycol dimethacrylate (TEGDMA) for use as a major component in a self-healing bonding resin. Methods TEGDMA nanocapsules were synthesized via interfacial polycondensation in a miniemulsion, and the TEGDMA nanocapsules were then characterized via Fourier-transform infrared (FTIR) spectrometer, field emission scanning electron microscopy (FESEM), and high-performance liquid chromatography (HPLC) to investigate the morphology, the average TEGDMA loading ( DL %), and encapsulation efficiency ( EE %). The mechanical property of dental adhesive with different concentrations (0, 3, 6, 9, and 12 wt%) of the TEGDMA nanocapsules were also measured, and the cytotoxicity was investigated using an MTT assay. Results FTIR confirmed that the TEGDMA nanocapsules were successfully synthesized. These nanocapsules showed a high drug load. The bond strength of the dental adhesive incorporated with 9 wt% TEGDMA nanocapsules was significantly higher compared with those of the other groups ( P < 0.001). Moreover, the biocompatibility of the dental adhesive was not affected by the incorporation of the TEGDMA nanocapsules. Conclusions The current study demonstrated the successful synthesis of TEGDMA nanocapsules, and the overall properties of the dental adhesive were not compromised. 1 Introduction The immediate bonding effectiveness of contemporary dentine adhesives has become favourable with the development in adhesive dentistry. However, the limited durability of dentine adhesives in vivo is still a major shortcoming. A number of factors affect the durability of adhesives in clinical applications, such as the degradation of exposed collagen fibrils, especially for etch and rinse adhesives, and the hydrolytic breakdown of adhesive monomers primarily for self-etch adhesives. Microcracking induced by thermal and mechanical fatigue is also a long-standing problem that affects the durability of adhesives. At present, methods of enhancing the durability primarily focus on the built-in dentine-associated matrix metalloproteinases (MMPs) inhibitors or the improvement of the bonding technique, such as ethanol wet-bonding. However, there are little studies focused on enhancing the durability by healing the microcracks. Tensile loading, fatigue loading, and thermal cycling can result in the formation of microcracks in dental adhesives. The nucleation of microcracks around weak links can propagate continuously, resulting in the degradation of mechanical properties, formation of pathways for the entry of liquids and bacteria, and complete material failure. Studies have indicated that the weak links in dental bonding adhesion are located in the hybrid layer, the quality of which is important to bond strength. Therefore, to improve the durability of dental adhesives, microcracks should be repaired before the integrity of the adhesive is destroyed. Scientists have recently proposed self-repair ideas that may increase the life expectancy of composite materials. The discovery of a self-healing resin based on the use of microcapsules is a research breakthrough. White et al. successfully synthesized a self-healing epoxy resin composite through the encapsulation of a healing agent within the microcapsules. These encapsulations were embedded with a catalyst capable of polymerizing the healing agent in a polymer matrix. The rationale for the self-healing design is that when cracks form in the matrix, they rupture the microcapsules, releasing the healing agent into the crack plane through capillary action. The healing agent then comes into contact with the catalyst, triggering polymerization, and finally bonds until the crack faces close. To date, the use of a self-repair bonding resin to improve the durability of dental adhesives is an open and intriguing approach. Meanwhile, the rapid development in nanoscale controlled drug delivery technology provides the possibility of a ‘smart’ material for the purposes of self-healing in the oral environment. Scientists have postulated that a chemical repair monomer encapsulated in a polymer can be an important factor in developing self-repair materials. Polyurethanes, an important class of polymers, are widely used in the medical field to encapsulate a wide range of active agents. The excellent physicomechanical properties, high flexibility, and relatively good biocompatibility of polyurethanes make them suitable shell materials for encapsulation and meet the requirements of self-healing nanocapsules. Moreover, triethylene glycol dimethacrylate (TEGDMA) is a frequently used monomer with low viscosity, making it a suitable candidate as a core material. Therefore, a hypothesis on the synthesis of nanocapsules encapsulated with an active monomer that exhibits the self-healing function of dental adhesives based on the aforementioned rationale is proposed. The primary objective of this pilot study was to investigate the synthesis and characterization of polyurethane nanocapsules containing TEGDMA via interfacial polycondensation in a miniemulsion for use as a major component in self-healing bonding resins. The mechanical properties and cytotoxicity were also determined to investigate the compatibility of TEGDMA nanocapsules with a commercial dental adhesive. 2 Materials and methods 2.1 Materials Isophorone diisocyanate (IPDI) and TEGDMA were obtained from Aldrich Chemical Company, Inc., USA. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma Chemical Company. Dulbecco's modified Eagle's medium (DMEM) and foetal bovine serum (FBS) were from Gibco Company. All other reagents were of analytical grade. 2.2 Fabrication of the TEGDMA nanocapsules The TEGDMA nanocapsules were encapsulated in polyurethane by interfacial polycondensation in miniemulsion of IPDI and 1,6-hexanediol as reported by Torini et al. Detailed process could be seen in our precious work and the brief procedure was showed in Fig. 1 . Fig. 1 Scheme of synthesis of TEGDMA-nanocapsule 0.88 g of SDS and 0.66 g HD were dissolved in 70 ml distilled water, and stirred by magnetic stirring apparatus at 300 rpm, 40 °C for 1 h. And then 6.66 g of TEGDMA and 2.22 g of IPDI were added and stirred for 10 min under the same conditions, after which high speed shear and ultrasonication were proceeded to form miniemulsion. At the end of ultrasonication, 5.91 g of 1,6-hexanediol solvent was slowly added to the miniemulsion. Through demulsification, centrifugation, rinse and lyophilisation, the TEGDMA nanocapsules were fabricated. 2.3 Morphological analysis The TEGDMA nanocapsules were dispersed in distilled water as mentioned above and then one droplet was dropped on tin foil. The sample was dried at room temperature and observed under FESEM (Sirion 200, FEI, Netherlands) at 15 kV. 2.4 TEGDMA loading and encapsulation efficiency Nanocapsules loaded with TEGDMA (10 mg) were dissolved in isopropanol followed by 30 min on ultrasounds. After extraction, it was diluted to volume with distilled water to 10 ml. Then 1 ml solution was extract and diluted to volume with mobile phase to 10 ml. The resulting solution was filtered through 0.45 μm PTFE membrane filters. And then the filtered solution (20 μl) was injected into a high performance liquid chromatographic (HPLC) apparatus. And the amount of TEGDMA entrapped was determined by HPLC. A ChromoQuest thermoquest France SA instrument was used with Agilent HC C-18 (5 μm, 250 mm × 4.6 mm). The mobile phase consisted of methyl cyanides and water (55:45). The flow rate was 1 ml/min. A volume of 20 μl loop was injected at temperature of 25 °C. TEGDMA was detected using UV spectrophotometer (Secomam, Anthelie, France) at the λ max value of 205 nm and results were quantified through the use of the equation derived from the slope of the standard curve prepared for TEGDMA ( R 2 = 0.9998) at 205 nm. These data were collected in triplicate, but the average values were considered in calculating % drug loading and encapsulation efficiency. These were measured using the following equations: DL%=WLWT×100% D L % = W L W T × 100 % DL is the TEGDMA loading; W L is the weight of TEGDMA in TEGDMA nanocapsules; and W T is the weight of TEGDMA nanocapsule powders. EE%=WLWa×100% E E % = W L W a × 100 % EE is the encapsulation efficiency; W a is the initial weight of TEGDMA taken for loading; and W L is the weight of TEGDMA in TEGDMA nanocapsules. 2.5 Polymer characterization Fourier transform infrared ray (FTIR) spectroscopy (Vertex 70, Bruker, Ettlingen, Germany) was performed using a potassium bromide disc on a spectrometer with a resolution of 4.00 cm −1 . Infrared spectra were recorded in the range of 500–4000 cm −1 to evaluate the molecular structure and phase clarification of blank nanocapsules and TEGDMA nanocapsules, respectively. 2.6 Micro-tensile bond testing Twenty freshly extracted human third molars were used in this test. The teeth were collected after the patients’ informed consent was obtained under a protocol approved by the Ethics Committee for Human Studies, School of Stomatology, Wuhan University. The occlusal one-third of the crown was removed by a water-cooled low-speed diamond saw (Isomet, Buehler, Evanston, IL, USA). A smear layer was created by abrading the dentine with 600 grit SiC paper under water. The prepared dentine specimens were randomly divided into five groups. Bonding procedures were performed on the treated occlusal surfaces of mid-coronal dentine with Prime & Bond NT (Dentsply De Trey, Germany) (NT) in the absence (control, Group A) or the presence of 3% (Group B), 6% (Group C), 9% (Group D) and 12% (Group E) of the TEGDMA nanocapsules. Composite buildups were performed using Charisma universal light curing Microglass ® composite (Heraeus Kulzer, Germany). For μTBS evaluation, composite-dentine beams of 0.8 mm 2 were stressed to failure at a crosshead speed of 1 mm/min ( n = 20 per group). The μTBS data were analysed by one-way ANOVA and Tukey's multiple comparison tests to determine any significance at 5% level. For the adhesive/dentine interfaces ultrastructure observation, intact specimens of each group were kept in advance. The adhesive/dentine interfaces and the surfaces of fractured specimens were sputter-coated with gold/palladium and examined with FESEM at 15 kV, respectively. 2.7 MTT assay Cytotoxicity of TEGDMA nanocapsules was identified measuring cell survival and proliferation in vitro by MTT assay, as previously reported. Mouse 3T3 fibroblasts were placed in a 96-well tissue culture dish at 1 × 10 4 cells/well. After 24 h of incubation, the medium was removed and cell monolayer was incubated in the absence of the control (Group 1) or the presence of extracts from Prime & Bond NT (Group 2), Prime & Bond NT + TEGDMA nanpcapsules (Group 3), and Prime & Bond NT + blank nanocapsules (Group 4) for 1 d, 3 d, and 5 d, respectively. The medium was replaced by 20 μL/well of a solution of MTT (0.5 mg/ml) in phosphate buffered saline (PBS), and the cells were incubated at 37 °C for 4 h in a 5% CO 2 atmosphere. The MTT solution was removed and replaced with 150 μL/well of DMSO and gently shaken until the crystals were dissolved. The optical density (OD) was immediately measured in a Bio-Rad ELISA reader at 490 nm. Each experiment was performed in triplicate. 3 Results 3.1 Morphology of TEGDMA nanocapsules The FESEM micrographs exhibit that the TEGDMA nanocapsules have a well-developed spherical shape and a smooth surface ( Fig. 2 A and B). No obvious coalescence phenomena and fragments are observed. Fig. 2 FESEM micrographs of nanocapsules (A and B): FESEM micrographs indicate that the TEGDMA nanocapsules are spherical with smooth and condense surface. 3.2 TEGDMA loading and encapsulation efficiency The mean TEGDMA loading ( DL %) and the average encapsulation efficiency ( EE %) are (47.7 ± 2.2) % and (90.5 ± 2.2) %, respectively. 3.3 Structure of TEGDMA nanocapsules FTIR spectrums of the blank nanocapsules and TEGDMA nanocapsules are exhibited in Fig. 3 . In Fig. 3 A, the spectrum shows absorption bands for the N–H vibration at 3370 cm −1 , the C O vibration of urethane at 1702 cm −1 , the urea carbonyl vibration at 1640 cm −1 , and the C–N vibration at 1561 cm −1 . The TEGDMA nanocapsule presents characteristic absorption peaks of TEGDMA at 1637 cm −1 (overlapped with the urea carbonyl absorption band at 1640 cm −1 ) for the C C group and at 1171 cm −1 for the C–O–C group ( Fig. 3 B). Fig. 3 FTIR spectrums of nanocapsules: A – blank nanocapsules, B – TEGDMA nanocapsules. (A) The spectrum shows absorption bands for the N–H vibration at 3370 cm −1 , the C O vibration of urethane at 1702 cm −1 , the urea carbonyl vibration at 1640 cm −1 , and the C–N vibration at 1561 cm −1 . (B) Strong peaks at 1171 cm −1 associated with TEGDMA can be observed. 3.4 Micro-tensile bond strength (μTBS) test Mean μTBS values obtained in all experimental groups are displayed in Fig. 4 . For Group A, the μTBS is 20.3 ± 4.9 MPa. For Group B, Group C, and Group E, the bond strengths are 19.2 ± 5.3 MPa, 17.8 ± 3.8 MPa, and 18.2 ± 3.2 MPa, respectively. The highest μTBS (25.8 ± 4.9 MPa) value is obtained from the adhesive contained 9 wt% nanocapsules ( P < 0.001). Typical fracture surfaces and adhesive/dentine interfaces are shown in Fig. 5 . Fig. 4 TBS values of five groups The μTBS of adhesive of Prime & Bond NT in the absence (control, Group A) or the presence of 3% (Group B), 6% (Group C), 9% (Group D) and 12% (Group E) of the TEGDMA nanocapsules (* P < 0.001, n = 20). Fig. 5 FESEM observations of the adhesive/dentine interfaces. (A and B) FESEM images of dentine (D) surface show that TEGDMA nanocapsule (black arrow) surrounded by adhesive are infiltrated into dentinal tubule (T) with resin tag (RT). (C and D) FESEM micrograph of the adhesive/dentine interface bonded with Prime & Bond NT or Prime & Bond NT incorporated with TEGDMA nanocapsules. The resin tags (white arrow) were longer and more regular as compared with the control. (E and F) Local magnification from (D) shows that TEGDMA nanocapsules (white arrows) could be notified on the surface of resin tag (RT). The rough and uneven surface indicates coverage of adhesive. 3.5 Cytotoxicity test The cytotoxicity data in this experiment are exhibited in Fig. 6 . The results demonstrate that extracts from NT with/without nanocapsules are cytotoxic with respect to control in the first 3 days under this experimental condition ( P < 0.05). However, eluates from Groups 1, 2, 3 and 4 are not significantly different from each other after 5 days ( P > 0.05). In Fig. 7 , it is observed that the cell number in Groups 2, 3, and 4 decreased compared with Group 1 during the first 3 days. When cultured for 5 d, there was no difference amongst the four groups. Fig. 6 OD values in MTT assay The proliferation of mouse 3T3 fibroblasts in the absence (Group 1) or the presence of extracts from Prime & Bond NT (Group 2), Prime & Bond NT + TEGDMA nanpcapsules (Group 3), and Prime & Bond NT + blank nanpcapsules (Group 4) for 1 d, 3 d, and 5 d, respectively (* P < 0.05). Fig. 7 Representative images of 3T3 fibroblasts cultured in the absence (Group 1) or the presence of extracts from Prime & Bond NT (Group 2), Prime & Bond NT + TEGDMA nanocapsules (Group 3), and Prime & Bond NT + blank nanocapsules (Group 4) for 1 d, 3 d and 5 d, respectively. 4 Discussion Providing simpler and faster-acting dental adhesives is necessary in the development of dental adhesives. However, optimizing the speed and efficiency should not jeopardize the quality and durability of dentine resin bonds. Considerable effort has been exerted to improve dentine bonding durability and stability. However, self-healing materials using nanocapsule technology have not yet been reported in the field of dental material research. In the current study, TEGDMA nanocapsules, which can be used as components for a self-healing bonding resin and may improve the durability of dentine bonding in the future, were successfully fabricated through interfacial polycondensation in miniemulsions. Fabrication approaches for nanocapsules are well-documented, including interfacial polycondensation, interfacial polymerization, interfacial precipitation, interfacial deposition, and self-assembly procedures. In view of the liquid core material used in the present study, we synthesized TEGDMA nanocapsules via interfacial polycondensation in miniemulsions. Miniemulsions are dispersions of relatively stable oil droplets in water prepared by shearing a system containing oil, water, surfactant and costabilizer. Appropriate preparation and formation of miniemulsions suppress the coalescence of droplets and Ostwald ripening, resulting in the formation of latex particles and initial droplets with similar sizes as well as in a relatively narrow size distribution. Results from a previous study on nanocapsule size and PDI are consistent with this theory. In Fig. 2 , TEGDMA nanocapsules are shown to have a smooth surface and a well-developed spherical shape, both of which can facilitate the dispersion of nanocapsules into the dental adhesive. The dense shell of a TEGDMA nanocapsule shown in Fig. 2 B can protect the activity of TEGDMA. Several studies have reported that high encapsulation efficiency nanoparticles were synthesized via miniemulsion polymerization. Both the drug loading and the encapsulation efficiency of the nanoparticles prepared via miniemulsion were higher than those obtained via emulsion. In the current study, the mean TEGDMA DL % and average EE % were (47.7 ± 2.2)% and (90.5 ± 2.2)%, respectively, which confirms the efficient encapsulation of TEGDMA in the nanocapsules. The relatively high TEGDMA DL % of the nanocapsules is important for the self-healing of dental adhesives. The FTIR results show that polyurethane nanocapsules containing TEGDMA were successfully synthesized. The primary principle of polyurethane formation through interfacial polycondensation is that isocyanates react with the hydroxyl groups of diols and amino-functional molecules via addition to the carbon–nitrogen double bond. Theoretically, this reaction should not trigger the polymerisation of TEGDMA, which is usually polymerized via free radical polymerization, during synthesis. In Fig. 3 A and B, characteristic vibrations (3370, 1702, 1640, and 1561 cm −1 ) associated with the polyurethane structure confirm the formation of polyurethane nanocapsules. In Fig. 3 B, although the characteristic absorption peaks of TEGDMA at 1637 cm −1 corresponding to the C C group overlapped with the urea carbonyl absorption band at 1640 cm −1 , a strong peak at 1171 cm −1 associated with TEGDMA was observed, which confirms that TEGDMA was successfully encapsulated in the nanocapsules. A dental adhesive or bonding resin is an intermediate resin layer between the composite resin and dentine. The primary role of this adhesive layer is to withstand the stress caused by the shrinkage of the composite during setting and to keep the restoration in place. In the current study, the microtensile bond test was selected to evaluate the performance of the dentine adhesive incorporated with TEGDMA nanocapsules. The microtensile bond strength of NT with 9 wt% TEGDMA nanocapsules was significantly higher (25.8 ± 4.9 MPa) compared with those of other groups ( P < 0.001), whereas the experimental adhesives containing 3, 6, and 12 wt% TEGDMA nanocapsules showed bond strengths similar to that of the control ( P > 0.05). The addition of particle fillers can result in an increase in fracture toughness via multiple phenomena, including crack pinning, crack bridging, microcracking, and crack deflection. The polyurethane shell material has a high level of flexibility and hardness; as the dimensions of the polyurethane capsules decrease, the load-bearing stress on the adhesive is reduced, thereby inhibiting crack formation and propagation. The spherical shape of the nanocapsules ( Fig. 2 ) provides smooth and rounded edges, resulting in an even distribution of stress throughout the adhesive. Additionally, a number of studies reported that the microtensile bond strength is significantly correlated with the ultimate microtensile strength of the adhesive resin. The bond strength is possibly correlated with strength-based mechanical properties, such as flexural strength and fracture toughness. Thus, the polyurethane nanocapsule concentration possibly affects the toughness of the adhesives. However, the redundancy of the nanocapsules may cause aggregation and consequently compromise the mechanical property. Therefore, a 9 wt% nanocapsule content may be the optimum concentration. Future studies should focus on the detailed evaluation of the effect of these new nanocapsules on the material properties of adhesives with different volume fractions. Changes in the adhesive properties under ageing conditions should also be investigated. FESEM images of the adhesive/dentine interfaces show different resin tag infiltrations ( Fig. 5 ). A number of studies found that a deeper penetration of the monomer into the demineralized dentine results in stronger micromechanical bonding. Evidence also indicates that bond strength relies on the quality of the hybrid layer and not on monomer infiltration. TEGDMA nanocapsules possibly aid monomer penetration without disturbing the hybrid layer. However, this finding remains to be verified. Monomers can be released from dental resin materials and thus result in cytotoxicity in vitro and show certain adverse effects in vivo. Previous studies also demonstrated that modern dentine adhesives, including Prime & Bond NT, exhibit cellular toxicity. In the current study, the result of the MTT assay was consistent with those of previous studies. However, the addition of TEGDMA nanocapsules to this commercial adhesive did not affect cellular toxicity, indicating that TEGDMA nanocapsules met the biocompatibility requirements in clinical practice. 5 Conclusions The synthesis and characterization of TEGDMA nanocapsules for use in a self-healing bonding resin were described in the current study. These nanocapsules showed not only a relatively narrow size distribution, high drug load, and good compatibility, but also potential mechanical enhancement of the bonding materials. The present study is a preliminary research on the application of a self-healing bonding resin in an oral environment. Further studies have to be conducted to achieve the self-healing of dental materials. For example, investigating an effective initiator system for TEGDMA and the rupture of nanocapsules are issues that need to be resolved. 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