Investigation of nano-SiO2 impact on mechanical and biocompatibility properties of cyanoacryalate based nanocomposites for dental application

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Investigation of nano SiO2 impact on mechanical and biocompatibility Properties of Cyanoacralate based nanocomposites for dental application Sima Afsharnezhad, Mehrdad Kashefi, Javad Behravan, Melika Ehtesham Gharaee, Mojtaba Meshkat, Khadijeh Shahrokh Abadi, Masoud Homayoni Tabrizi www.elsevier.com/locate/ijadhadh

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S0143-7496(14)00130-4 http://dx.doi.org/10.1016/j.ijadhadh.2014.06.004 JAAD1551

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International Journal of Adhesion & Adhesives

Received date: 17 December 2013 Accepted date: 23 April 2014 Cite this article as: Sima Afsharnezhad, Mehrdad Kashefi, Javad Behravan, Melika Ehtesham Gharaee, Mojtaba Meshkat, Khadijeh Shahrokh Abadi, Masoud Homayoni Tabrizi, Investigation of nano SiO2 impact on mechanical and biocompatibility Properties of Cyanoacralate based nanocomposites for dental application, International Journal of Adhesion & Adhesives, http://dx.doi.org/ 10.1016/j.ijadhadh.2014.06.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Investigation of Nano SiO2 impact on Mechanical and Biocompatibility Properties of Cyanoacralate based Nanocomposites for Dental Application

Sima Afsharnezhad1*, Mehrdad Kashefi2, Javad Behravan3, Melika Ehtesham Gharaee3, Mojtaba Meshkat4, Khadijeh Shahrokh Abadi5, Masoud Homayoni Tabrizi5 1- Department of Biochemistry, Mashhad Branch, Islamic Azad University, Mashhad, Iran. 2- Department of Materials Science and Metallurgical Engineering, Faculty of Engineering, Ferdowsi University of Mashhad, Mashhad, Iran. 3- Biotechnology Research Center, Department of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran. 4- Department of Biostatistics, Mashhad Branch, Islamic Azad University, Mashhad, Iran. 5- Department of Biology, Mashhad Branch, Islamic Azad University, Mashhad, Iran.

*Corresponding Author:  Assistant Professor, Department of Biochemistry, School of Medicine P O Box 91775-1365 Mashhad Iran

Tel: (+98-511-2250041) Fax: (+98‐511‐2250048)  E‐mail: [email protected] 

ABSTRACT

1   

Although cyanoacrylate glues are widely used in medicine, cyanoacrylate based nanocomposites have been recently suggested for dental restorative/filling applications. In the present research, SiO2 nanoparticles were used as filler for development of a novel dental nanocomposite base on alkoxy-ethyl-cyanoacrylate. The nanocomposite samples filled with different levels of nano-sized SiO2 (wt %) were made the mechanical properties were evaluated and comparisons were made with the neat cyanoacrylate. The hardness and wear behavior of the samples were measured using Vickers hardness and pin-on-disk tester, respectively. The wear mechanism of the samples was also evaluated using scanning electron microscopy (SEM). Furthermore, cell biocompatibility of the samples using MTT and LDH assays as well as inflammatory cytokine expression interleukin-6 (IL-6) from L929 cells were investigated. The results showed that an increase in nano-sized SiO2 content improves hardness and wear resistance of the cyanoacrylate based nanocomposites and changes the wear mechanism from adhesive to abrasive. The results of cytotoxicity analysis showed a significant reduction in cell viability and IL-6 produced from the samples-exposed L929 cells compared with untreated control cells. Moreover, increasing in nano SiO2 powder content caused a decrease in the released formaldehyde. Keywords: Cyanoacrylate, nano-sized SiO2, nanocomposite, biocompatibility, mechanical properties

1. Introduction Solvent free cyanoacrylate (CA) adhesives [CH2=C (CN) COOR] are polar and linear molecules, which are based on acrylic monomers and can be polymerized by both free radical and anionic mechanisms, but the latter holds much greater interest [1]. The anionic reaction is inhibited at pH < 5.5 [2]. Lewis acids are appropriate inhibitors which include sulphur dioxide [5], carboxylic acids, trichloroacetic, picric, paratoluene sulfonic acid [3] and sulfones [1]. Prior to polymerization, neutralization of the acids takes place. Any practical, weak electron donor base such as moisture on the surface of a substrate, can initiate the polymerization reaction. Basic chemicals as sodium carbonate, phosphates, amines and pyridine [1, 3] and caffeine [4] can be used to neutralize the stabilizer and so accelerate polymerization. Due to their high adhesion through covalent bond formation, CAs have been widely used in medicine for tissue bonding and dental applications [5]. They can be used in dentistry for tooth cavity treatment [6], tooth pit, fissure sealants [7] and desensitizers [8]. Using fillers can effectively improve the mechanical and teribiological properties of the cements such as wear and hardness [9, 10]. Although, most widely adopted bonding systems in density use micro- and nano-composite resins, using the composites have some limitations since the properties usually involve compromises in mechanical properties [11, 12]. Mechanical and tribological parameters such as hardness and wear properties not only affect the overall mechanical performance of the dental materials but they would also affect the surrounding tissues in the mouth [13, 14] as well as produce biological active particles which in turn could stimulate inflammatory responses [15]. Research also indicates that CA 2   

compound is unstable and could release formaldehyde under the environment conditions, causing cell toxicity [16]. Hence highly stable and biocompatible, nano powders such as SiO2 [17] can be used to improve properties of CAs. Few studies have been reported on the toxicity of reinforced CAs [13]. Therefore, the present research was aimed at investigating some of the mechanical properties as well as biocompatibility of CA reinforced with nano-sized SiO2 powders (CA/SiO2 nano-composite). The nanocomposite may be considered as potential for dental restorative/filling applications.

2. Materials and Methods 2.1. Nanocomposite Synthesis Alkoxy-ethyl-CA (Loctite 460, Henkel) with a viscosity of 40 mPa.s and polygonal silica (10-20 nm) powders (Nanolin, China) with a purity of 99% % was used as matrix and filler, respectively. The low viscosity and low blooming effect of the alkoxy-ethyl-CA make it a good candidate as a matrix for dental nanocomposite. To break down any unwanted agglomeration the nano powder was initially milled by mixer mill device (Retsich MM400, Germany) for 10 minutes prior to mixing. In order to prevent polymerization of alkoxy-ethyl-CA by surface moisture and any other electron donors, para-toluene solfunic acid (PTSA) was utilized. Neutralization should take place before polymerization [18, 19]; hence, caffeine (Merck) was used as an activator. To prepare nano composite samples, first, 1 wt% of acid was solved in the alkoxy-ethylCA prior to mixing with the SiO2 nano powder to act as an inhibitor. Then, different weight percents of SiO2 nano powders

(9, 11, 13 wt %) were mixed with alkoxy-ethyl-CA using magnetic stirrer. Due to high

viscosity and molding difficulties, 13% was the highest weight percentage of the powder that could be incorporated into the mixture. Finally, 0.5 wt% of caffeine dissolved in the mixtures to trigger polymerization and the resulted nanocomposites were molded in cylindrical shape and allowed to cure. The cured samples were machined to final cylindrical shape of 3 mm diameter and 8 mm high. The flow chart of fabrication process and the specifications of resulted samples are summarized in Fig. 1 and Table 1, respectively. In order to examine the chemical structure of the nanocomposite Fourier transform infrared 3   

(FTIR) spectra were recorded. FT-IR studies were carried out on samples dispersed in KBr discs using a Shimadzu IR Prestinge-21 (20 scans per spectrum at 4 cm-1 resolution). 2.2. Mechanical Analysis of synthesized nanocomposites In the present research, the pin-on-disk wear test [20] was utilized. Each sample was attached to a removable holder with alkoxy-ethyl-CA glue. Al2O3 abrasive plate with diameter of 25 mm (Dedeco, #309-0009) and artificial Fusayama Meyer saliva [21] were used as the abrasive plate and media, respectively. Xiaoqiang et al. [22] expressed that the chewing stress on occlusal surface of teeth varies between 0.8 - 1.75 MPa. Thus, wear tests were done under two minimum and maximum forces (5.6 N and 12.3 N) to achieve the corresponding stresses. After each 2500 orbits, both saliva and aluminum oxide abrasive plates were replaced. Samples weights were measured with the accuracy of ±0.0001g before and after testing and wear rate was calculated by the following formula; Wear Rate = ΔW/L. where ΔW is the difference of weight measurement before and after 2500 orbits and L (in meter) is the distance for the rotations and calculated as follows; L = 2500 × 2πR = 157 m For each formulation (according to Table 1), 5 samples were tested and the average wear rate values reported. Samples were also investigated using scanning electron microscope (LEO 1450 VP, Germany) to characterize wear mechanism. For hardness measurements, samples were first cold mounted by acrylic resin. The Vickers hardness, Hv, of the specimens was measured by micro-hardness test device (MicroMet 4, Buehler) using the following formula [23]. Hv =

1.854 P d2

Where P is the applied load (kgf) and d is the mean indentation diagonal length (mm). For each formulation 15 indentations were done. 2.3. Biological analysis 2.3.1. Preparation of sample extract 4   

Cylindrical samples (neat alkoxy-ethyl-CA and alkoxy-ethyl-CA/ SiO2 nano composites containing 9, 11 and 13 wt % of SiO2) were made following ISO-10993:12 guideline [24]. The ratio of surface area to volume of the samples and their mass to the extract volume were kept at 6 cm2/ml and 0.1 g/ml, respectively. Then, the samples were sterilized by ethylene oxide (EtO) and placed in the extraction vehicle for 72 h at 37 °C while shaking constantly. The extracts were then filtrated (0.45 µm pore size) to eliminate the possibility of presence of solid particles. The indirect contact test was performed as follows: The extracts were separated by decantation, being the designated 100 % extract. Released extracts were not suspension and diluted. 2.3.2. Cell Culture L929 mouse fibroblast line (ATCC CCL-1) was purchased from Pasteur Institute of Iran (cell Bank). The cells in medium RPMI1640 (Gibco, USA) supplemented with 10%(W/V) fetal calf serum (FCS), 50 unit/ml penicillin and 50 µg/ml streptomycin (all from Invitrogen Gibco), at 37°C in a 5% CO2-humidified atmosphere were cultured. Viability of cells throughout the experiment was always >95% as determined by trypan blue assay. 2 × 104 cells from log phase cultures were seeded in 200 μl of RPMI medium supplemented with 10% fetal bovine serum per well of 96-well flat-bottom culture plates (Nunc, Denmark). The cells were incubated with the extracts for a defined time (24, 48, and 72 hours). The cells which were grown on polystyrene for cell cultures, (without added any extract), were used as negative control.

2.3.3. MTT assay The cytotoxic effect of extract samples on L929 cells was assessed by the MTT [3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyl tetrazolium bromide] assay [25]. Briefly, About 2×106 cells/mL/well was incubated in 96 well plates for 24, 48 and 72 hours. 5 mg mL-1 MTT dye (Sigma, St. Louis, MO) in PBS was prepared freshly before each test and filtered through a 0.2 μm filter to sterilize and remove un-dissolved particles. For each 200 μL growth medium, 20μL MTT solutions was added and incubated for 4 hours. After removing the growth medium, resulted crystals were dissolved in 150 μL DMSO (Dimethyl sulfoxide) and 25 μL glycine buffer by shaking microplates for 2-3 minutes. The absorbance of formazan dye was recorded at 570 nm using ELISA plate reader (Biotek, USA). This assay was performed in triplicate, with two independent replicates. 2.3.4. LDH release 5   

Cellular cytotoxicity assessment of extract samples-exposed L929 cells was determined by lactate dehydrogenase (LDH) release using non-radioactive cytotoxicity assays (Roche, Germany). After incubating the L929 cells with the sample extracts for 24, 48 and 72 h exposure time at 37 °C in a humidified atmosphere with 5% CO2, 2 × 104 cells/ml from samples were centrifuged at 250×g for 10 min. To determine LDH activity in the supernatants 100µl reaction mixture was added to each well and incubated for 30 min at 20ºC. The cells treated with 1% Triton-X 100 (500 µl) were used as high control groups. The absorbance of the samples at 492 nm measured using an ELISA reader (Biotek, USA). All experiments were performed in triplicate in test. To determine the percentage of cytotoxicity, the resulting values were substituted in the following equation.   Where, low control is LDH activity released from the untreated-normal cells and high control is the maximum releasable LDH activity in the cells. 2.3.5. Inflammatory cytokine expression IL-6 kit (Bender Med System, Vienna, Austria) was used to determine L929 cells cytokine level. 100 µl culture supernatants were added to the wells following exposure for 24 h to the extracts. According to the manufacturer’s instruction, each well of ELISA plate was filled with anti-IL-6-monoclonal antibody, and then samples of IL-6 were conjugated with biotin and were added to wells and kept for 2 hours at room temperature. The samples were rinsed with distilled water in order to eliminate unbounded compounds. Finally, streptavidin - HRP was added to the conjugated biotin- IL-6. Rising were carried out at room temperature for one hour and samples were assessed at 450 nm. Untreated cells served as controls and experiments were performed in triplicate. 2.3.6. Formaldehyde concentration The measurement of the formaldehyde concentration was performed by a visible absorption spectrometry. A 4ml from each extract solution was placed into 25 ml glass-stoppered flasks then 0.1 ml 1% chromotropic acid added to the flask and mixed. Then, 6 ml H2SO4 (2N) was added slowly to the flask. The solutions were heated in 95°C for 15 min. After cooling the solution to the room temperature (2-3 hours), the samples absorbance was read at 580 nm. The results for each sample were averaged (n = 3). 3. Statistical evaluation of data The Kolmogorov-Smirnov with Lilliefors correction test was used to evaluate the normality of samples. Groups were compared using the analysis of variance test and Tukey post hoc test. All data were presented as means±SD. P-value <0.05 was considered to be statistically significant. 6   

3. Results 4.1. FTIR analysis The infrared spectrums of neat alkoxy-ethyl-CA and the optimum nano composite containing 13 wt % SiO2 is shown in Fig. 2. The solid state vibration in SiO2 was related to the absorption bands at 440, 800 cm–1 and 1064 cm-1 ,as well as ,1410 cm-1 for δas Si−CH3 and 880 cm-1 for δ

drocking

Si−C in −Si(CH3)2 [26]. More

vibrations can be seen for carbonyl groups ,triply bonded carbon (−C≡C−) and corresponding covalent vibration (≡C−H) which appeared at 1720 , 2250 and 3300 cm-1, respectively. The peaks attributed to N-H2 and two different CH groups were also observed at 664, 1250 and 3000 cm-1, respectively [27]. New bands between Si and alkoxy-ethyl-CA were formed. This could result in subsequent reduction in the movement of the polymer chains, which in turn, could be the cause for differences in physical and mechanical properties of the nanocomposites compared to the neat alkoxy-ethyl-CA.

4.2. Mechanical measurements Table 2 presents the results of the micro hardness test. As it can be seen, the addition of nano-SiO2 powder led to an increase in the composite hardness. As Fig 3indicates, adding more SiO2 resulted in less wear for both maximum and minimum forces (12.3 N and 5.6 N). Furthermore, addition of the wear mechanism from adhesive to abrasive (Fig 4).

4.3. Biological analysis 4.3.1. MTT assay

7   

nanopowder alter

As shown in Fig. 5, the cell viability is directly proportional functions of SiO2 wt% in the nano composites (r2 = 0.73). According to Tukey post-hoc test, there was no significant difference (P=0.934) in cytotoxicity between AE -0 and nanocomposites at any exposure times of the extracts up to 72 h, except AE -13.

4.3.2. LDH assay One-way analysis of variance (ANOVA) tests revealed that AE -13 had the least toxicity and the highest toxicity was measured for AE -0 sample in comparison with control group (P=0.001). Furthermore, the cellular cytotoxicity was significantly decreased with increasing SiO2 wt% in the alkoxy-ethylCA/nanocomposites (P=0.01, Fig. 5).

4.4.3. Inflammatory cytokine expression Cytokine IL-6 expression data from the extract-exposed L929 cells highlights significantly increased in inflammatory conditions (P=0.001) compared with untreated control cells. Besides, Mann-Whitney test revealed that there was no significant difference (P=0.19) in IL-6 level between all the samples, although AE -13 was induced slightly less IL-6 expression than other nanocomposites (Fig. 6).

4.3.4. Formaldehyde Concentration Formaldehyde levels in the alkoxy-ethyl-CA extract and nanocomposites are illustrated in Fig. 7. The lowest in formaldehyde level was related to AE -13 and the highest were observed in AE-0 sample. Furthermore, there was only significant difference between AE -0 and AE -13 samples (P= 0.02).

5. Discussion Although cyanoacrylate base dental nanocomposite was studied by few researchers investigating different properties of the nanocomposite, there is a gap for evaluating mechanical and biological properties on the same cyanoacrylate based dental cement. 8   

5.1. Reinforcing effect of alkoxy-ethyl-CA with nano SiO2 particles on mechanical properties Most dental nanocomposites experience load during fabrication and /or chewing. Hence, their hardness and wear properties are among the most important mechanical characteristics of dental materials. Tomlinson et al. studied hardness development of dental nanocomposites based on ethyl cyanoacrylate matrix using glass and hydroxyapatite [28]. They concluded hardness of the nanocomposite was a function of powder/ cyanoacrylate ratio and the type of the filler. As Table 2 shows, the addition of nano-SiO2 powder improves nanocomposite hardness. It can be observed that the hardness of nano composite improved sharply by addition of SiO2 powders (9 %) .The further increase in hardness by adding more nano powder was gradual. Generally, improving hardness of the nanocomposite is a function of the hardness of the filler powder particles themselves, their effect on the degree of cyanoacrylate polymerization, its bond strength with matrix and ratio of the mixture components [29]. Wear is a complex tribological phenomenon resulted from many contributing parameters such as contact load, distance and the surrounding media (saliva, food) [30].Therefore, an in vitro experimental design was used in order to study some basic wear properties of the fabricated nano composite. As Figure 3 indicates, an increase in powder percentage had a significant effect on improving the wear resistance of neat cyanoacrylate. Besides, comparing SEM images of the worn samples under maximum load (Fig. 4) revealed a change in wear mechanism from adhesive (AE -0) to abrasive (nanocomposites) wear. Although the wear mechanism remains abrasive for all nanocomposites, adding more nano powder causes the worn morphologies of the nanocomposites become less coarse. This could be as the result of presence of harder phase which impede the propagation of cracks in the soft matrix. Reinforcing effect of alkoxy-ethyl-CA with nano SiO2 particles on cytotoxicity propertiesThe main concern about dental CAs applications is the possible release of substances that may be harmful to the oral mucosa [31, 32]. In the present study, the hypothesis has been taken that adding silica nanoparticles to alkoxy‐ethyl‐ CA glue improves its mechanical properties as a dental material as well as affecting its biocompatibility. The nanocomposites used in the experimental study solidified quickly in moisture so it could not be distributed properly in medium and failed to give accurate results while contacting directly to cells (data are not shown). 9   

Besides, the viscosity of the nanocomposites was very high which made it impossible for performing the direct contact test. Therefore, cytotoxic effect of the released substances from the nanocomposites was performed using extract dilution test and toxic extracts of the cured samples on L929 mouse fibroblast tissue were investigated. L929 cell line was selected because it is mostly used for short term studies of cytotoxicity studies in dental materials [33,34]. Using both direct and extract dilution tests, laboratory-based research show that CAs are toxic on cell cultures [35, 36]. Similar results were also obtained on various commercial CA grades [31, 32 and 37]. Although, a few studies [38] have been conducted on the toxicity of alkoxy‐ethyl‐ CA but there is no study on reinforced alkoxy-ethyl-CA. Mizrahi et al. showed that alkoxy‐ethyl‐CAs, especially their derived hexose, have less toxicity compared to even octhyl-CA (Dermabond) on Hela cells. According to their results, cell viability was 30% for alkoxy-ethyl-CA extract, 50% and 40% for hexoxy-CA, and Dermabond, respectively [38]. Our  data  demonstrated  that, the mean and standard deviation of the L929 cell viability for neat alkoxy‐ethyl‐CA (AE ‐0) were 36.18±6.8 while the corresponding data for AE -13 were 64.35 ± 10.61 (Fig. 6). One interesting finding which could be deduced from the results is that adding silica nono particle to alkoxy‐ethyl‐CA  caused  a  change  in  the cytotoxicity mechanism (Fig. 7). It can be explained as follows; in LDH test, which is based on cell membrane damage [39], the minimum damage and the lowest toxicity was observed for AE -13 (the highest percentage of nano SiO2 used in the research). However, MTT assay results showed that the cell viability decreased after 48 and 72 hours exposure. Although decreasing in cell numbers at 24hrs is likely related to metabolic activity. On the other hand, LDH results showed no toxic effect on mouse fibroblasts at any exposure times. Therefore, it can be concluded that changes in L929 viability (compared to control cells) may be due to the different mechanisms such as effects on receptors, inflammatory reactions, signaling pathways or regulation of cell activity which cause  cell  viability  reduction. The most important components which is involved in inflammatory of pulp and periapical, are cytokines TNF-α, IL-1, IL-6 and IL-8 of [40], but in the present study we used interleukin 6 as it has both pro-inflammatory and anti-inflammatory properties, synergistic effect on IL-1 and TNF-α [41] as well as enjoying easier tracking in compare with the other ones. In the present study, all extracts induced 10   

IL-6 release from L929 cell (Fig. 6). This finding would suggest that the synthesized nanocomposites were able to initiate inflammatory by cytokine release from cells. Schmalz et al. demonstrated that molecules important in the initiation of inflammation like PGE2 or IL-6 and IL-8 were released from human oral tissue culture models after exposure to compounds of dental materials [42]. As CAs release formaldehyde [43], the concentration of alkoxy-ethyl-CA in each of the extracts was also evaluated (Fig. 7). The result showed that AE-0 had the highest and AE-13 the lowest levels of formaldehyde. The formation of new bands in nano composites (Fig. 2) and increasing the stability of molecular structure of alkoxy-ethyl-CA could be a reason for the reduction in formaldehyde release with increasing wt% of filler in nono composites. The exact mechanism of action of formaldehyde toxicity is not clear, but it is known cytotoxic, mutagenic and pro allergenic potential, which is mediated for example by protein–DNA, adducts [44]. It was suggested that CAs could release other substances such as Alkyl Alcohols [38]. In general, the mode of Alkyl Alcohols action is disruption of biological membrane function  [45]. According to LDH result, our data also demonstrated the L929 cell viability reduced in exposure of all samples especially for AE -0 (Fig. 5). However, this issue emphasizes on the need for more studies on the substances release from CAs and their mechanism in in-vitro and in-vivo conditions. 6. Conclusion The effect of reinforcing alkoxy-ethyl-CA with nano-sized SiO2 powders on mechanical properties as well as biocompatibility and antibacterial was studied and the following results were obtained. 1- The presence of SiO2 nanoparticles improved the hardness and reduced the wear rate of alkoxy-ethyl-CA 2-

Addition of SiO2 nanoparticles causes a change in wear mechanism from adhesive to abrasive.

3- Increasing wt% of nano SiO2 powders resulted in a reduction of levels of cytotoxicity, the released formaldehyde from the nanocomposites and stimulation of IL-6 in L929 cells. Acknowledgments The authors thank Dr. T. Abdolahi for helpful discussions. This work was supported by the Islamic Azad University of Mashhad Branch (grants No A-1357). 11   

References: [1]. A. Ludwig, The Use of mucoadhesive polymers in ocular drug delivery, Advanced Drug Delivery Reviews, 57 (2005) 1595-1639. [2]. S.H Ng, J.B Hull, J.L Henshall, Machining of novel alumina-cyanoacrylate green ceramic compacts, Journal of Materials Processing Technology, 157 ( 2006) 299-305. [3]. C. Birkinshaw, M. Buggy, A. O'Neill, Injection molding and extrusion. International application published under the patent cooperation treaty (PCT), Chem J, (1996). [4]. I. Bravo-Osuna, C. Vauthier, A. Farabollini, G.F. Palmieri, G. Ponchel, Mechanism of chitosan and thiolated chitosan-Poly (Isobutyl Cyanoacrylate) core-shell nanoparticles, Biomaterials, 28 (2007) 2233-43. [5]. M. Perez, I. Fernandez, D. Marquez, R.M. Bretana, Use of N-butyl-2-cyanoacrylate in oral surgery: biological and clinical evaluation, Artif Organs, 24 (2000) 241–3. [6]. E.L Herod, Use of cyanoacrylate adhesives in dentistry, J Canad Dent Assoc, 56 (1990) 56: 331–4. [7]. J.L Lage-Marques, R Conti, J.H Antoniazzi, The use of Histoacryl in endodontics, Braz Dent J, 3(1993) 95–8. [8]. L.W Ripa, Sealants revisited: an update of the effectiveness of pit-and-fissure sealants, Caries Res, 27 (1993) 77– 82. [9]. E. Sancaktar,, Adhesives and Sealants, Design: Fatigue and Fracture Mechanics, Engineered Materials Handbook Volume 3: Adhesives and Sealants, H. F. Brinson, ASM International Materials Park Ohio,1990, pp. 501-520 [10]. J.S. Ridgway, J.B. Hull, C.R Gentle, A prime approach for the moulding of conduit ceramic parts, Journal of Materials Processing Technology, 133 (2003)181-8. [11]. O.N. Klenovich, A.M. Vetrova, Filled cyanoacrylate adhesive compositions, Polymer Science Series C, 49 (2007) 50-51. [12]. S.P. Passos, A.P. Freitas, S. Jumaily, J.R. Santos, M.C Maria., A.S. Rizkalla , et al, Comparison of Mechanical Properties of Five Commercial Dental Core Build-Up Materials, Compend Contin Educ Dent,43( 2013)62-68. [13]. G.S. Lee, S.B. Lee, D.Y. Lee, K.J. Park, S.O. Kim, K.N. Kim, et al, Physical and adhesive properties of cyanoacrylate-based beta-TCP Composites, Key engineering materials,1 (2007) 419-422. [14]. P.P. Binon, Implants and Components, Int. J. Oral Max, 15 (2000)76–94. [15]. M. Torabinejad, T.R. Ford, H.R. Abedi, S.P.Kariyawasam, H.M. Tang, Tissue reaction to implanted root-end filling materials in the tibia and mandible of guinea pigs, Journal of Endodontics, 24(1998) 468-71. [16]. Y.C. Tseng , Y. Tabata , S.H. Hyon , Y. Ikada , In vitro toxicity test of 2-cyanoacrylate polymers by cell culture method, J Biomed Mater Res, 24 (1990)1355-67. [17]. M.A. Malvindi , V. Brunetti , G. Vecchio , A. Galeone , R. Cingolani , P.P. Pompa , SiO2 nanoparticles biocompatibility and their potential for gene delivery and silencing, Nanoscale, 21 (2012) 486-95. [18]. S. Guzmán-Armstrong, R.J. Mitchell, Surface coating and leakage of dentin-bonded resin composite restorations, J Dent, 30(2002)113-8. [19]. Zhang. Y, Zhu. S, Yin. L, Qian. F, Tang. C, Yin. C, Preparation, characterization and biocompatibility of poly (ethylene glycol) poly (n- buthyl cyanoacrylate) nanocapsules with oil core via miniemulsion polymerization, European Polymer Journal, 44 (2008) 1654–1661. [20]. R. Jacobi, H.T. Shillinbury, M.G. Dencanson, A comparison of the abrasiveness of six ceramic surfaces and gold, Journal of Prosthetic Dentistry, 66(1991)303-9. [21]. J.M. Meyer, J.N. Nally, Influence of artificial saliva on the corrosion of dental alloys, Journal of Dental Research, 54 (1975) 678-81. [22]. H.U. Xiaqiang, P.M. Marquis, A.C. Shortall, Two body in vitro wear study of some current dental composites and amalgam, Journal of Prosthetic Dentistry, 82 (1999)214-20. [23]. R.L. Sakaguchi, M. Cross, W.H. Douglas, A simple model of crack propagation in dental restorations, Dental Material, 8 (1992)131-6. [24]. ISO 10993-10, Biological Evaluation of Medical Devices, Part 10, Tests for Irritation and Delayed-Type Hypersensitivity, 2002. [25]. H. Gunzler, H.M. Heise. IR-Spektroskopie, 2nd ed. VCH, Weinheim, Gemany, 1996, pp. 56-60. [26]. N.J. Marshall, C.J. Goodwin and S.J. Holt, A critical assessment of the use of microculture tetrazolium assays to measure cell growth and function, Growth Regulation, 5(1995) 69–84. [27]. G. Socrates, Infrared Characteristic Group Frequencies, J. Wiley & sons, Chichester, UK, 1994, pp. 120 -131.

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[28]. S.K. Tomlinson, P. Brookbank, O.R. Ghita, M. Barbour, K.E. Evans, Novel dental cements using a new resin (cyanoacrylate) and new coated fillers (hydroxyapatiote and TiO2), 13th European Conference of Composite Materials (ECCM13), Sweden, 2008. [29]. S. K. Tomlinson, R. O. Ghita, M. Robert, Hooper, Kenneth E. Evans. Monomer conversion and hardness of novel dental cements based on ethyl cyanoacrylate, Dental materials, 23(2007) 799–806. [30]. Z.R. Zhou, J. Zheng, Tribology of dental materials: a review, J. Phys. D: Appl. Phys, 41 (2008)1-22. [31]. V. Thumwanit, U. Kedjarune, Cytotoxicity of polymerized commercial cyanoacrylate adhesive on cultured human oral fibroblasts, Australian Dental Journal, 44 (1999) 248–52, [32]. T. Poskus, M. Rosana, S. Lima, Cytotoxicity of current adhesive systems: in vitro testing on cell culture of L929 and balb/c3T3 fibroblasts, Rev Odonto, 11(2009) 129-133. [33]. M. Kaga, M. Noda, J.L. Ferracane, W. Nakamura, H. Oguchi, H. Sano, The in-vitro cytotoxicity of

elutes from dentin bonding resins and their effects on tyrosine phosphorylation of L929 cells, Dent Mater, 17 (2001) 333-9. [34]. E. Koliniotou-Koubia, P. Dionysopoulos, E.A. Koulaouzidou, A.H. Kortsaris, Y. Papadogiannis, Invitro cytotoxicity of six dentin bonding agents, J Oral Rehabil, 28 (2001) 971-5. [35]. G. Ciapetti ,  S. Stea ,  E. Cenni , A. Sudanese , D. Marraro , A. Toni ,  A. Pizzoferrato , Cytotoxicity testing of

cyanoacrylates using direct contact assay on cell cultures, Biomaterials, 15 (1994)63-7. [36]. G. Ciapetti , S. Stea , E. Cenni , A. Sudanese , D. Marraro , A. Toni , A. Pizzoferrato , Toxicity of cyanoacrylates in vitro using extract dilution assay on cell cultures, Biomaterials, 15 (1994) 92-6. [37]. V. Thumwanit, U. Kedjarune, Cytotoxicity of polymerized commercial Cyanoacrylate adhesive on cultured human oral fibroblasts, Australian Dental J, 44 (2005)248-52. [38]. B. Mizrahi, C.F. Stefanescu, C. Yang, M.W. Lawlor, D. Ko, R. Langer, et al, Elasticity and safety of alkoxyethyl cyanoacrylate tissue adhesives, Acta Biomaterialia, 7 (2011)3150–57. [39]. D.M. Kendig, J.B. Tarloff, Inactivation of lactate dehydrogenase by several chemicals: implications for in vitro toxicology studies, Toxicol In Vitro, 21 (2007)125–32. [40]. P.M. Preshaw, J.J. Taylor, How has research into cytokine interactions and their role in driving immune

responses impacted our understanding of periodontitis? Journal of Clinical Periodontology, 38 (2012) 60-84. [41]. K. Brebner, S. Hayley, R. Zacharko, Z. Merali, H. Anisman, Synergistic effects of interleukin-1β,

interleukin-6, and tumor necrosis factor-α: central monoamine, corticosterone, and behavioral Variations, Neuropsychopharmacology, 22 (2000) 566-80. [42]. G. Schmalz, , H. Schweikl, K.A. Hiller, Release of prostaglandin E2, IL-6 and IL-8 from human oral epithelial culture models after exposure to compounds of dental materials, Eur J Oral Sci, 108 (2000)442-48. [43]. J.L. Cameron, S.C. Woodward, E.J. Pulaski, H.K. Sleeman, G. Brandes, R.K. Kulkarni, F. Leonard,The degradation of cyanoacrylate tissue adhesive, J Surgery, 58 (1965)424-30. [44]. C.E. Vaca, J.A. Nilsson, J.L. Fang, R.C. Grafstrom, Formation of DNA adducts in human buccal epithelial cells exposed to acetaldehyde and methylglyoxal in vitro, Chemico Biological Interactions, 108 (1983)197–208. [45]. V. Wezel, A. Opperhuizen, Narcosis Due to Environmental Pollutants in Aquatic Organisms: Residue-Based Toxicity, Mechanisms, and Membrane Burdens, Critical Reviews in Toxicology, 25 (1995) 255-279.

Table 1. Designation and composition of synthesized nanocomposites. Sample

Designation

Polymer content (wt %)

13   

Acid content (wt %)

Caffeine content (wt %)

SiO2 content (wt %)

Alkoxy-ECA/0%SiO2 Alkoxy-ECA/9%SiO2 Alkoxy-ECA/11%SiO2 Alkoxy-ECA/13%SiO2

AE-0 AE-9 AE-11 AE-13

97.5 88.5 86.5 84.5

1 1 1 1

1.5 1.5 1.5 1.5

0 9 11 13

Table 2. Microhardness test data for neat cyanoacrylate and synthesized nanocomposites. Sample

Hardness (HV)

AE-0

14.3

AE-9

16.13

AE-11

16.80

AE-13

18.06

  Table 3.Data extracted from TGA and DSC curves for neat cyanoacrylate and the optimum nanocomposite. Sample

Released heat (J/g)

Temperature rise (°C)

T50% (°C)

AE-0

202.64

16

232.24

AE-13

139.73

2

246.12

 

14   

Fig.1. Flow chart of steps in manufacturing the nanocomposite samples.

Fig. 2. FTIR spectra of (A) neat alkoxy-ethyl-CA (AE -0) and (B) optimum nano composites containing 13  wt %  of SiO2(AE -13).

  Fig. 3. Wear test results in Fusayama Meyer artificial saliva for both maximum and minimum forces (12.3 N and 5.6 N). 15   

  Fig. 4. SEM images of the worn samples. Arrow indicates change in wear mechanism from adhesive for neat alkoxyethyl-CA (AE -0) to abrasive for nano composites containing 9 wt % (AE-9), 11 wt % (AE -11) and 13 wt % (AE -13) of  SiO2.

     

  

 

Fig. 5. L929 cell viability after exposure to neat alkoxy-ethyl-CA (AE -0) and nano composites containing 9 wt % (AE-9), 11 wt % (AE -11) and 13 wt % (AE -13) of SiO2 (A) MTT assay of L929 cells with or without extract contact with test materials after incubation 24 h, 48 h and 72 h. (B) LDH assay of same. Data are presented as mean ± SD (n=3 per group). Asterisks denote statistical difference from unexposed cells (control). *P < 0.05, **P < 0.01, ***P < 0.001. 16   

Fig.6. Boxplot analysis (with standard error bars) of IL-6 expression (in pg ml -1) from untreated control L929 cells following 24h exposure to the extract of neat alkoxy-ethyl-CA (AE -0) and nano composites  containing 9 wt % (AE-9), 11 wt % (AE -11) and 13 wt % (AE -13) of SiO2.

 

  Fig.7. Boxplot analysis (with standard error bars) of) formaldehyde concentration (µmol) in extract of neat alkoxy-ethyl-CA (AE -0) and nano composites  containing  9 wt  %  (AE-9),  11  wt  % (AE -11) and  13  wt  %  (AE -13) of SiO2.

17