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Biomechanical properties of nano-TiO2 addition to a medical silicone elastomer: The effect of artificial ageing
journal of dentistry 42 (2014) 475–483
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Biomechanical properties of nano-TiO2 addition to a medical silicone elastomer: The effect of artificial ageing Linlin Wang a,1, Qi Liu a,1, Dongdong Jing b, Shanyu Zhou a, Longquan Shao a,* a b
Department of Stomatology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China Department of Pharmacy, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
article info
abstract
Article history:
Objectives: The aim of this study was to evaluate the effect of TiO2 nanoparticles on the
Received 13 October 2013
mechanical and anti-ageing properties of a medical silicone elastomer and to assess the
Received in revised form
biocompatibility of this novel combination.
29 December 2013
Methods: TiO2 (P25, Degussa, Germany) nanoparticles were mixed with the silicone elasto-
Accepted 3 January 2014
mer (MDX4-4210, Dow Corning, USA) at 2%, 4%, and 6% (w/w) using silicone fluid as diluent (Q7-9180, Dow Corning, USA). Blank silicone elastomer served as the control material. The physical properties and biocompatibility of the composites were examined. The tensile
Keywords:
strength was tested for 0% and 6% (w/w) before and after artificial ageing. SEM analysis was
Nano-TiO2
performed.
Silicone elastomer
Results: TiO2 nanoparticles improved the tensile strength and Shore A hardness of the
Biomechanical properties
silicone elastomer (P < 0.05). However, a decrease in the elongation at break and tear
Artificial ageing
strength was found for the 6% (w/w) composite (P < 0.05). All the ageing methods had no effect on the tensile strength of the 6% (w/w) composite (P > 0.05), but thermal ageing significantly decreased the tensile strength of the control group (P < 0.05). Cellular viability assays indicated that the composite exhibited biocompatibility. Conclusions: We obtained a promising restorative material which yields favourable physical and anti-ageing properties and is biocompatible in our in vitro cellular studies. # 2014 Elsevier Ltd. All rights reserved.
1.
Introduction
Defects of the maxillofacial area can cause embarrassment for patients. The first choice of treatment is the fitting of a prosthetic following ablative surgery, which can improve the patient’s quality of life.1,2 Silicone elastomer is a promising functional material used for the correction of maxillofacial defects. However, this material does have drawbacks, since natural or outdoor weathering of silicone elastomers can
induce significant changes in its physical and mechanical properties.3 In addition, the conventional disinfection method can accelerate ageing of the facial epitheses.4 Ultimately, this means that the patient must undergo several prosthetic replacements. In this regard, there have been several previous studies on the longevity of facial prostheses, with earlier reports indicating that the average prosthetic lifetime is 6–12 months.5,40 With the continued improvement of clinical care, the demands from both doctors and patients regarding the
* Corresponding author. Tel.: +86 02062787153. E-mail address: [email protected] (L. Shao). 1 These authors contributed equally to this work and should be considered co-first authors. 0300-5712/$ – see front matter # 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jdent.2014.01.002
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performance of silicone elastomers are increasing. Unfortunately, this demand has not been satisfied by the conventional silicone elastomers used in facial epitheses. More recent modifications to silicone elastomers have improved their properties. For example, Liu et al.6 added hollow microspheres into the MDX4-4210 elastomer to decrease density and improve flexibility, and showed that a 5% (v/v) hollow microsphere composite yields a light, soft, flexible, and biocompatible maxillofacial prosthetic material. Han et al.7 found that the incorporation of nano-oxide (Ti, Zn, or Ce) improved the mechanical properties of the A-2186 elastomer (FACTOR II, USA). However, to the best of our knowledge there have been no reports describing improvements to the anti-ageing properties of facial epitheses used in the oral environment. Titanium dioxide (nano-TiO2) nanoparticles have been proposed as a reinforcing material for addition to dental composites. Elsaka et al.8 showed that incorporation of nanoTiO2 nanoparticles into conventional glass-ionomer (GI) improved its mechanical and antibacterial properties. Mixing of zirconia nanoparticles with a primer or an adhesive increased the bond strength of the adhesive system through reinforcement of the adhesive layer and resin tags.9 On the contrary, the addition of TiO2 and SiO2 nanoparticles to poly(methyl methacrylate) acrylic resins adversely impacts the flexural strength, and this effect is directly related to the concentration of nanoparticles.10 Biocompatibility is the ability of a material to function optimally in a particular application, while having minimal impact on the host response. This is a critical feature that distinguishes materials used in biological applications from other high-tech materials.11,12 The biological and toxicological properties of dental materials are the most important criteria on which clinicians base their decisions for facial epitheses selection.13 The MTT assay is a reliable and sensitive method for detecting cell growth and survival.14 While the studies outlined above indicate that silicone elastomer is the preferred material for maxillofacial epitheses, the rapid ageing of facial epitheses in a service environment frequently underlies why such facial epitheses ultimately fail. With this in mind, the aim of the present study was to improve the performance of silicone elastomer properties. Specifically, we used three artificial ageing tests that simulate the oral environment to determine the effect of nanoparticle additives on the tensile strength of elastomers.
2.
Materials and methods
Test samples were obtained by blending 2%, 4%, and 6% (w/w) proportions of TiO2 (P25, Degussa, Germany) nanoparticles and the medical silicone elastomer (MDX4-4210, DOW Corning, USA) in Teflon molds. Thinners (Q7, DOW Corning, USA) were added at a mass ratio of 1:0.8 to the total mass of MDX44210, and TiO2 nanoparticles were weighed using a balance with an accuracy of 0.0001 g (BSA234S, Sartorius, Germany). Subsequently, the TiO2 nanoparticles and curing agent (maintained at a SE:curing agent mass ratio of 10:1) were mixed with the silicone elastomer using a bull magnetic stirrer (HJ-6, Jintan Fuhua Electric Apparatus Co Ltd, China) in a 50-ml glass beaker. Blank silicone elastomer served as the control
group. Air bubbles in the mixture were eliminated by 30 min incubation in a vacuum oven (SHZ-DIII, Nanhe Zhicheng Science and Technology Development Co Ltd, China). Finally, the mixture was allowed to cure for 2 h at room temperature to allow for gas escape. Further curing was then carried out in a high temperature chamber (YLCD-8000P, KELONG, China) at 60 8C for 4 h. The specimens were stored at 23 1 8C for 24 h before testing.
2.1.
Tensile strength and Elongation at break
Thirty-six type 2 dumbbell-shaped specimens were prepared using a silicone elastomer cutter (N = 36, n = 9) (Fig. 1). The tensile strength was measured according to ISO 37:2005 standard on a servo control computerized tensile testing machine (TH-8201S, Tuobo Machinery Co Ltd, China) at a crosshead speed of 500 mm/min. Tensile strength, TS (MPa), was calculated as follows: F TS ¼ Wb where F is the peak force (N), W is the specimen width of narrow parallel portion (mm), and b is the specimen thickness (mm). The elongation at break, Eb (%), was calculated using the following equation: ðL L0 Þ Eb ¼ 100 b L0 where Lb is the test length at break (mm) and L0 is the initial test length (mm).
2.2.
Tear strength
Twenty-four crescent-shaped specimens were prepared using a silicone elastomer cutter (N = 24, n = 6) (Fig. 2). The tear strength was measured based on the ISO 34-1:2004 standard using a servo control computerized tensile testing machine at a crosshead speed of 500 mm/min. Tear strength, TS (KN/m), was calculated according to the following formula: F TS ¼ d where F is the tearing force (N) and d is the specimen thickness (mm).
2.3.
Shore A hardness
Twelve bar-shaped specimens were prepared in a Teflon mold (70 mm 40 mm 6 mm) (N = 12, n = 3). Each sample was
Fig. 1 – Sample size of tensile strength (mm): A = 152 mm, B = 55 mm, C = 25 mm, D = 13 mm, R = 13 mm.
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The values of tensile strength before and after ageing were recorded.
2.5.3.
Stress fatigue
Hertzian contact testing was performed using an experimental system of high-precision biological materials (Electro Force 3510, BOSE, USA). The specimens were submitted to stress fatigue for 66,700, 133,300, 200,000, and 266,700 cycles at room temperature. The load was applied through a stainless steel plate (10 mm 8 mm 7 mm) at a load force of 75 N, and a speed of 72 times/min. The values of tensile strength before and after ageing were tested.
2.6. Fig. 2 – Sample size of tear strength (mm): A I 110 mm, B = 25 W 0.5 mm, C = 68 W 1.5 mm, D = 45 W 0.2 mm, E = 10.5 W 0.05 mm, R1 = 7.5 W 0.5, R2 = 43 W 0.2, R3 = 12.5 W 0.1, R4 = 9 W 0.2.
read at five points. The surfaces of the specimen were cleaned by alcohol before testing. Shore A hardness was measured by a Shore A durometer (LX-A SCLEROMETER, Shangshen, China) based on the ISO 7619-2008 standard. The mean values were recorded.
2.4.
Scanning electron microscopy
The specimens were sputtered with a thin gold layer using a sputter coater (HITACHI, E-1010, Japan). Subsequently, the morphology of the fractured specimens of silicone rubber was observed using a scanning electron microscope (SEM) (HITACHI, S-3700N, Japan).
2.5.
Anti-ageing test
2.5.1.
Thermal ageing
The thermal ageing tests were run in a high temperature chamber (accurate to 1 8C) (YLCD-8000P, KELONG, China). The 6% (w/w) composite and the blank silicone elastomer were subjected to thermal ageing according to the ISO 188:2007 standard. The ageing procedures were conducted at four temperature (50 8C, 100 8C, 150 8C and 200 8C) for 72 h. The sample was suspended for free state to the stainless grids. The distance between each of the two samples was at least 5 mm, and the distance between the sample and the walls of the chamber was at least 70 mm. The values of tensile strength before and after ageing were recorded.
2.5.2.
UV ageing
The UV ageing experiment was performed in the QUVWeathering Tester (QUV/Spray, Q-La, USA). Conditions were 4 h UV irradiation with a power of 0.68 W/m2/nm at 50 3 8C and 4 h water condensate at 50 3 8C. The specimens were submitted to the UV ageing process based on the ASTM D 458701 standard. The 6% (w/w) composites were exposed to UV irradiation by applying a lamp type UVA-340 for 24, 48 or 72 h.
Cytotoxicity testing (MTT assay)
Circular diaphragms (14 mm diameter, 2 mm thickness) of the 6% composite were prepared. All samples were treated using high-pressure steam sterilization. The samples were placed in a medium (RPMI-1640, GIBCO, USA) with 10% foetal bovine serum (16000-044, GIBCO, USA) at 37 8C for 24 h (specific surface area 3 cm2/mL, ISO 10993-5:2009), filtered through 0.22-mm cellulose acetate filters (Millipore; Sigma) prior to being placed in the refrigerator at 4 8C. The specimens were divided into experimental group, negative control group and positive control group. The positive control group was a medium containing 6.4% phenol solution. Cells were seeded in 96-well cell culture plates at 5.0 103 NIH/3T3 mouse fibroblasts cells mL1 per well. The multiwell plates were incubated at 37 8C, 5% CO2 for 24 h. Each group consisted of 5 replicate wells. The culture medium was aspirated from the wells and equal volumes (100 mL per well) of the extracts were added for a further 24 h. In the negative control wells, 100 mL DMEM was added. In positive control wells, 100 mL DMEM with 0.64% phenol solution was added. Then the multiwell plates were incubated at 37 8C, 5% CO2 for 24 h, 48 h or 72 h, after which each well received 20 mL of MTT (5 mg/mL) for 4 h. MTT was then removed and 100 mL DMSO was added to each well, with shaking for 10 min. Subsequently, the absorbance at 570 nm was measured using a UV– visible spectrophotometer (Molecular Devices, USA). Survival rates of the negative controls were set to represent 100%.
2.7.
Statistical analysis
Data of physical and mechanical tests were analyzed using one-way analysis of variance (ANOVA). Two-way classification ANOVA was utilized in analysis of the MTT assay (a = 0.05). Dunnett’s T3 post hoc test was used to determine differences between any of the groups, with a significance level of P < 0.05.
3.
Results
3.1.
Tensile and tear properties
Table 1 demonstrates the mean tensile and tear strength values. Data of tensile strength showed an insignificant increase for the 2% and 4% (w/w) composites (P > 0.05), followed by significantly increased tensile strength at
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Table 1 – The physical properties of SE incorporated with TiO2 nanoparticles. Group
Tensile strength (MPa)
SE-control SE-2% (w/w) TiO2 SE-4% (w/w) TiO2 SE-6% (w/w) TiO2
2.65 2.80 3.01 3.29
a
(0.09) (0.25) a (0.35)a,b (0.40) b
Elongation at Break (%) 203.23 254.28 192.83 142.15
a
(31.20) (20.14) b (16.46) a (14.63) d
Tear strength (MPa) 10.21 12.58 10.50 6.03
Shore A hardness (MPa)
a
(1.19) (1.91) a (0.77) a (0.530) b
25.07 28.67 30.53 31.97
(1.130) a (0.84) b (1.13) c (1.65) d
The different superscripts mean significant difference (P < 0.05).
concentrations of the 6% (w/w) group (P < 0.05). There was a notably significant increase in elongation at break for the 2% (w/w) composite (P < 0.05). There was no significant difference between the control and 4% composite (P > 0.05). However, the elongation at break significantly decreased in the 6% (w/w) composite (P < 0.05). There was an insignificant trend of increase followed by decline in the tear strength for the 2% and 4% (w/w) composites (P > 0.05). However, the mixing of 6% (w/w) TiO2 nanoparticles with silicone elastomer significantly decreased the tear strength (P < 0.05).
3.2.
Shore A hardness
Shore A hardness significantly increased with increasing concentration of TiO2 nanoparticles incorporated into the silicone elastomer (P < 0.05).
3.3.
3.4.
Ageing
3.4.1.
Thermal ageing
Fig. 4 presents the mean tensile strength values for the 0% and 6% (w/w) groups before and after thermal ageing. The effect of thermal ageing on the tensile strength of 6% (w/w) was not statistically significant (P > 0.05). However, the 0% (w/w) group presented a significant reduction of tensile strength at 200 8C (P < 0.05).
UV ageing
Fig. 5 shows the mean tensile strength values for 0% and 6% (w/w) groups before and after UV ageing. In this test, the 6% (w/w) group yielded higher tensile strength values than the 0% (w/w) group, irrespective of the ageing interval. The 0% and 6% (w/w) groups did not significantly decrease the effects of UV ageing (P > 0.05), although the 0% (w/w) group showed a trend of declined ageing as exposure time increased.
3.4.3.
3.5.
MTT assay
Table 2 shows that the cell survival rate of the 6% (w/w) group was comparable to that of the negative control group (P > 0.05). However, the positive control had a significant reduction in cell survival rate (P < 0.05). Table 3 shows the effect of incubation periods on the cytotoxicity towards NIH3T3 cells. Specifically, increasing the incubation period from 24 to 72 h leads to significant differences in viability (P < 0.05).
4.
Discussion
4.1.
Tensile and tear properties
SEM examination
A representative SEM photomicrograph of the fracture surface of the concentration difference of the TiO2 group revealed that 2% TiO2 nanoparticles were distributed inside the matrix without agglomeration; however, as the concentration of TiO2 nanoparticles increased to 4% and 6% (w/w), the phenomenon of nano-TiO2 agglomeration was apparent in the composite (Fig. 3).
3.4.2.
statistically significant in the control group and the 6% group (P > 0.05).
Stress fatigue
Fig. 6 presents mean tensile strength values for the 0% and 6% (w/w) groups before and after stress fatigue. The effect of accelerated stress cycling on the tensile strength was not
Prosthetics are subjected to tensile stress from every direction when in use, and therefore increasing tensile strength can prolong the useful lifetime of the prosthetic itself.15 Here, we found that a 6% (w/w) concentration of TiO2 nanoparticles mixed with silicone elastomer significantly improved tensile strength compared to the control group. This increase in tensile strength may be due to the dispersal of nanoparticles in the continuous phase of the silicone elastomer, which leads to increased cross-sectional area and force as well as a crosslinked structure formation of the composite material.16 Thus, we provide direct evidence that filling silicon elastomers with TiO2 nanoparticles is associated with strengthening of the material.
Table 2 – Cell viability of groups. Group
OD570 nm
RGR (%)
Level
Experimental group Negative control group Positive control group
0.49 (0.05) a 0.45 (0.11) a 0.09 (0.01) b
104.26 100 19.15
0 0 4
Note: The different superscripts mean significant difference (P < 0.05).
Table 3 – OD values of incubation periods. Incubation periods 24 h 48 h 72 h
N 15 15 15
OD570 nm a
0.30 0.36 b 0.38 b
SE 0.16 0.19 0.22
Different uppercase letters indicate statistical difference (P < 0.05).
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Fig. 3 – SE filled with TiO2 nanoparticles (a, b, c, d, 40T) and (b, d, f, h, 3500T): (a and b) 0% (w/w), (c and d) 2% (w/w), (e and f) 4% (w/w), (g and h) 6% (w/w).
The muscle actions during chewing, talking and laughing cause the remodelling of facial organs such as eyes, mouth and nose. Thus, the ideal facial epitheses should have a certain degree of flexibility, which can not only avoid the damage of facial epitheses, but also give the facial epitheses a more
natural appearance.17 Since the 2% concentrations of nanoTiO2 increase the elongation at break of the silicone elastomer, this may improve the deformation characteristics of facial epitheses. This may be due to the high specific surface area of nano-TiO2, which is likely to reinforce the contact area and the
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Fig. 4 – Tensile strength versus ageing temperature at 50 8C, 100 8C, 150 8C, 200 8C.
extent of binding.18 Studies show that the interaction between the fine particulate filler and matrix can increase the elongation at break of the filling system.19 However, the concentration of nanoparticles in silicone elastomers at 4% and 6% exhibited a certain degree of agglomeration, in which the molecular chains were fixed more firmly around the nanoparticles, weakening the interaction with the silicone elastomer.20 Kulik et al.21 and Chatterjee22 suggested that the high modulus characteristics of nanoparticles can decrease the elastic modulus of the filling material. However, it is imperative that facial epitheses used for the repair of soft tissue defects retain elasticity. The tear strength is also an important indicator of quality for evaluating facial epitheses. Clinically, the ideal shape of facial epitheses is gradually thinner from the middle to the surrounding. The edge is the weakest part of the facial epitheses and is susceptible to damage from external forces.23 Therefore, tear strength is an important factor to ensure longterm and stable reparative effects of facial epitheses. In this study, increases in the tear strength of the silicone elastomer were insignificant in the presence of 2% and 4% (w/w) TiO2 nanoparticles. This may be consistent with previous studies that examined tensile strength. For example, one study reported that nano-sizes of ZnO nanoparticles may result in a wider range of particle distribution attributable to the increased junctions in the composite due to the nano-size effect. The statement means nanoparticles have the nano-size effect, which could improve the mechanical properties of materials.24 However, 6% (w/w) TiO2 nanoparticles weaken the
Fig. 6 – Tensile strength versus stress fatigue frequency for 66,700 times, 133,300 times, 200,000 times, 266,700 times.
tear strength of silicone elastomers compared to the blank silicone elastomer alone. This could be due to the agglomeration of nanoparticles, resulting in poor interfacial bonding, which might force cracks not only along the cutting, but also down into the micro-defects of the nano-TiO2 filler/elastomer matrix. Usually, TiO2 nanoparticles can bond to polysiloxane. Thus, when the amount of nano-TiO2 is increased, there may be an inadequate amount of polysiloxane to link the nanoparticles effectively, which would lead to a decrease in the interfacial bonding in the nano-TiO2 silicone elastomer material.25
4.2.
We found that the Shore A hardness of blank silicon elastomer was lower than that of nano-TiO2 silicone elastomer. This could be due to dispersing of nanoparticles in the silicone elastomer, which increases the crosslink density, thereby leading to increased hardness.26 Another explanation could be that the nanoparticles affect the elastic modulus of the silicone elastomer.27 The modulus of elasticity of silicone elastomer is proportional to the Shore A hardness.28 The Shore A hardness is a measure of the texture of silicone elastomer, with a value between 25 and 35 indicating similarity with soft tissue, according to the defect area.29 In this study, the Shore A hardness of nano-TiO2 silicone elastomer composite was broadly in the range of ideal values, which might meet the hardness requirements of specific anatomical sites, such as maxillary, orbital, nasal and ear tissues. At these sites, the skin is very thin and the bone and cartilage are very close to the surface, creating the need for different texture requirements on facial epitheses compared to other soft tissues.30
4.3.
Fig. 5 – Tensile strength versus UV-ageing time for 24 h, 48 h, 72 h.
Shore A hardness
Ageing
Prosthetic materials must maintain long-term stability in order to function optimally. The effect of ageing can lead to a decline in physical and mechanical properties of the silicone elastomer, shortening the life of facial epitheses, which ultimately leads to replacement. Therefore, anti-ageing properties are important factors for improving facial epitheses stability. The prosthetic material must also be somewhat able to resist external forces, particularly in the context of oral function. Tensile strength is the load that silicone elastomer
journal of dentistry 42 (2014) 475–483
can bear upon stretching prior to fracture, and dictates the maximum capacity to resist tensile failure.30 The study chose tensile strength as an evaluation index for anti-ageing properties, simulating an artificial ageing environment in vitro to test the performance of silicone elastomer. Hatamleh et al.31 showed that silicone elastomer stored in a dark environment for 6 months presented slightly decreased initial performance, but a sharp decline in properties when exposed to the surrounding environment. The common factors that affect the performance of silicone elastomer materials are temperature, light, and mechanical force. Laboratory trials have attempted to recreate an oral environment, although these do have limitations. One clear difference is that these laboratory ageing processes are not carried out in the presence of saliva. Another difference is that many ageing processes do not occur at the same time in the lab test, and so further clinical research is required.32 As is well known, temperature accelerates oxidationreduction reactions within the internal structure of silicone elastomers. The internal facial epitheses may produce deformation and heat during chewing, swallowing and laughing, which can further promote the ageing of the silicone elastomer. Accordingly, laboratory simulations show that thermal ageing for 72 h at 250 8C can simulate 3 years of clinical facial epitheses usage.33 Additionally, facial epitheses can be affected by temperature, chemical substances and mechanical friction. This is why, in the current study, we performed thermal ageing for 72 h at 50, 100, 150, and 200 8C, which is equal to 0.5, 1, 1.5 and 2 years of clinical usage. After thermal ageing, the 6% group of nano-TiO2 silicone elastomers exhibited better anti-heat ageing performance than the other groups, but the tensile strength of the control group significantly decreased as temperature increased. Another group showed that increased temperature will result in hydrolytic scission at siloxane bonds of the main chain, and oxidation decomposition reactions of the side chain of silicone elastomers.34 Nano-TiO2 is a heat-resistant additive and can thus significantly improve the cross-linking reaction temperature of polysiloxane side groups, which in turn improves the heat ageing properties of the silicone elastomer.22 Furthermore, the thermal oxidation stability of the silicone elastomer is inversely proportional to the size of the additive particles.35 Illumination can engender ageing of silicone elastomers, with shorter wavelengths of higher energy (such as UV) producing the greatest destructive effects.3 Currently there are no estimates of the relationship between experimental UV irradiation of prosthetic materials and clinical usage. Because of the lack of such a connection, UV-ageing time intervals were decided by an association of the thermo-cycling process and clinical usage. The thermal ageing process took 86 h to conduct 2000 thermo-cycles, which is equivalent to 2 years of clinical facial epitheses usage.36,37 Considering the above, UV ageing was carried out for 24, 48, and 72 h, which is roughly equivalent to 0.5, 1 and 1.5 years of clinical wear, respectively. Fig. 5 indicates that the 6% (w/w) group composite and the control group had insignificant differences through the UV ageing process. However, the tensile strength of the control group tended to decrease over the course of UV ageing. This suggests that UV ageing time can be increased in order to draw
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more meaningful conclusions in further studies. Shen et al. showed that gamma rays alter the chemical structure and cross-linking density of silicone elastomer composites.38 Similarly, the duration of UV ageing engenders a proportional increase in hydrolytic reactions and accelerates the scission of the main chain in the elastomer.34,22,35 It has been found that nano-TiO2 has a strong ability to resist UV rays. Indeed, the nanoparticles can not only absorb, but also reflect and scatter UV rays due to their refractive index and optical activity.3 Therefore, modification of silicone elastomer material with TiO2 nanoparticles may also improve resistance to environmental UV irradiation. Minami et al.39 showed that 66,700 rounds of cyclic loading correspond to a 0.5-year period of intra-oral conditions. Therefore, in our study we performed 66,700, 133,300, 200,000, and 266,700 rounds in order to simulate 0.5, 1, 1.5 and 2 years of clinical usage. It also reported a bite force of 75 N, which must be the maximum for repetitive loading cycles. The values in Fig. 6 show that the control group and the 6% (w/w) composite group presented a more satisfactory elastic behaviour in tensile strength tests after stress fatigue. Stress fatigue is essentially generated by the combined effects of mechanical force, oxidation, and temperature. When a force acts on the silicone elastomer material, inhomogeneities of the internal structural network can result in the uneven distribution of stress and fracture of local molecular chains. Fong et al.40 showed that when the silicone elastomer was stressed with small amplitude and low frequency forces under conditions of high temperature and oxygen, the valence force of the molecular chain weakened, and the oxidation of silicone elastomer components increased. The oral cavity is a complex microbial environment filled with saliva, which may also generate local pockets of oxidation, and thus decrease the performance of facial epitheses. Stress fatigue has less impact on silicone elastomer prosthetic material than thermal ageing.
4.4.
MTT assay
In 2009, Miroslawa et al.41 studied the in vitro and in vivo cytotoxicity of silicone elastomer with TiO2 nanoparticles. They revealed that silicone elastomer containing TiO2 nanoparticles was non-toxic by a simple propidium iodide (PI) exclusion test using mouse fibroblast 3T3 cell line. Although the type of silicones we used here differs from that used by Miroslawa et al., our findings were essentially consistent with their study. Other researchers have studied the biocompatibility of room-temperature silicone elastomers using the MTT assay,42,43 and concluded that cell survival in the presence of silicone elastomers was affected by incubation periods. In our current study, we found that cell survival increased proportionally with the length of the incubation period. However, we did not demonstrate stability of the nano-TiO2 silicone elastomer in the context of biological reactions. For the special physical and chemical character, nano-TiO2 has been utilized in many fields and brings great progress in people’s life. However a new problem has turned to be the focus of people’s attention, that is, whether nano-TiO2 may bring about potential health risks to the organism. Srivastava et al.44 found that nano-TiO2 induces toxicity mechanism on
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oxidative stress, genotoxicity and apoptosis of human lung cancer cell line A549. Srivastava et al.45 suggested that the skin can be a potential tissue for photo-oxidative injury. Nano-TiO2 displayed prominent photocatalytic activity by stimulating the formation of protein tyrosine nitration, and the optimum condition for the reaction was around physiological pH. However, a review based on the current weight of evidence of all available data shows that the in vitro genotoxic and photogenotoxic profiles of these nano-structured metal oxides are of no consequence to human health.46 Therefore, the biocompatibility of epitheses material still needs further study prior to clinical application. Finally, since nano-TiO2 has photocatalytic activity, this novel composite material may have a potential application as a kind of antibacterial facial epitheses. Further research is certainly warranted to determine whether other properties of this composite make it suitable for long-lasting and antibacterial facial epitheses.
5.
Conclusion
1. A novel maxillofacial silicone elastomer mixed with TiO2 nanoparticles was synthesized. 2. In summary, silicone elastomer filled with 2% (w/w) TiO2 nanoparticles results in a material with improved physical properties for the maxillofacial prostheses. However, the elongation at break and the tear strength of the 6% (w/w) composite were significantly compromised. 3. The TiO2 silicone elastomer composite conferred the ideal values of Shore A hardness. 4. The TiO2 nanoparticles improve the anti-thermal ageing properties of silicone elastomer. 5. Cytotoxicity tests showed that the silicone elastomer filled with nano-TiO2 particles had short-term biocompatibility.
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Acknowledgments This work was supported by National Natural Science Foundation (50973045 and 31070857), Foundation for Highlevel Talents in Higher Education of Guangdong, China (201168), Research Fund for the Doctoral Program of Higher Education of China (20104433120006), and Foundation of President of NanFang Hospital (2013B013).
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biomaterials. Journal of Materials Science: Materials in Medicine 2010;21:1403–11. Goiato MC, Pesqueira AA, Santos DMD, Dekon SFDC. Evaluation of hardness and surface roughness of two maxillofacial silicones following disinfection. Brazilian Oral Research 2009;23:49–53. Chen MS, Udagama A, Drane JB. Evaluation of facial prostheses for head and neck cancer patients. Journal of Prosthetic Dentistry 1981;46:538–44. Liu Q, Shao LQ, Xiang HF, Xiang HF, Zhen D, Zhao N, et al. Biomechanical characterization of a low density silicone elastomer filled with hollow microspheres for maxillofacial prostheses. Journal of Biomaterials Science Polymer Edition 2013;24:1378–90. Han Y, Kiat-amnuay S, Powers JM, Zhao Y. Effect of nanooxide concentration on the mechanical properties of a maxillofacial silicone elastomer. Journal of Prosthetic Dentistry 2008;100:465–73. Elsaka SE, Hamouda IM, Swain MV. Titanium dioxide nanoparticles addition to a conventional glass-ionomer restorative: influence on physical and antibacterial properties. Journal of Dentistry 2011;39:589–98. Lohbauer U, Wagner A, Belli R, Stoetzel C, Hilpert A, Kurland HD, et al. Zirconia nanoparticles prepared by laser vaporization as fillers for dental adhesives. Acta Biomaterialia 2010;6:4539–46. Ahmad Sodagar DDS, Khalil S, Shahroudi AS. The effect of TiO2 and SiO2 nanoparticles on flexural strength of poly (methyl methacrylate) acrylic resins. Journal of Prosthodontic Research 2013;57:15–9. Liang SL, Cook WD, Thouas GA, Chen QZ. The mechanical characteristics and in vitro biocompatibility of poly (glycerol sebacate)-bioglass elastomeric composites. Biomaterials 2010;31:8516–29. Chalk J, Richmond BG, Ross CF, Strait DS, Wright BW, Spencer MA, et al. A finite element analysis of masticatory stress hypotheses. American Journal of Physical Anthropology 2011;145:1–10. Liu H, Zhang L, Yang D, Ning N, Yu YC, Yao L, et al. A new kind of electro-active polymer composite composed of silicone elastomer and polyethylene glycol. Journal of Physics D: Applied Physics 2012;45:485303. Ahmed B, Butt AM, Hussain M, Amin M, Yazdanie N. Rehabilitation of nose using silicone based maxillofacial prosthesis. Journal of the College of Physicians and Surgeons Pakistan 2010;20:65–7. Chen J, Kou S, Poon C. Hydration and properties of nanoTiO2 blended cement composites. Cement and Concrete Composites 2012;34:642–9. Li Q, Xia YY, Tang JC, Wang RY, Bei CY, Zeng Y. In vitro and in vivo biocompatibility investigation of diamond-like carbon coated nickel-titanium shape memory alloy. Artificial Cells Blood Substitutes and Biotechnology 2011;39: 137–42. Geurtsen W. Biocompatibility of root canal filling materials. Australian Endodontic Journal 2001;27:12–21. Ashraf H, Taherian A, Kerdar AN. Evaluation of cytotoxicity of two root canal filling materials by MTT assay. Australian Endodontic Journal 2010;36:24–8. Kong SM, Mariatti M, Busfield JJC. Effects of types of fillers and filler loading on the properties of silicone rubber composites. Journal of Reinforced Plastics and Composites 2011;30:1087–96. Zhou W, Qi S, Tu C, Zhao H, Wang C, Kou J. Effect of the particle size of Al2O3 on the properties of filled heatconductive silicone rubber. Journal of Applied Polymer Science 2007;104:1312–8. Kulik VM, Boiko AV, Bardakhanov SP, Park H, Chun HH, Lee I. Viscoelastic properties of silicone rubber with admixture
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35. Balachandran K, Venckatesh R, Sivaraj R. Synthesis of nano TiO2–SiO2 composite using sol–gel method: effect on size, surface morphology and thermal stability. International Journal of Engineering Science and Technology 2010;2: 3695–700. 36. Takahashi JMFK, Consani RLX, Henriques GEP, de Arruda No´bilo MA, Mesquita MF. Effect of accelerated aging on permanent deformation and tensile bond strength of autopolymerizing soft denture liners. Journal of Prosthodontics 2011;20:200–4. 37. Ribeiro Pinto JR, Mesquita MF, de Arruda No´bilo MA, Elias Pessanha Henriques G. Evaluation of varying amounts of thermal cycling on bond strength and permanent deformation of two resilient denture liners. Journal of Prosthetic Dentistry 2004;92:288–93. 38. Diao S, Zhang S, Yang Z, Feng S, Zhang C, Wang Z, et al. Effect of tetraphenylphenyl-modified fumed silica on silicone rubber radiation resistance. Journal of Applied Polymer Science 2011;120:2440–7. 39. Minami H, Suzuki S, Minesaki Y, Kurashige H, Tanaka T. In vitro evaluation of the effect of thermal and mechanical fatigues on the bonding of an autopolymerizing soft denture liner to denture base materials using different primers. Journal of Prosthodontics 2008;17:392–400. 40. Fong N, Simmons A, Poole-Warren L. Elastomeric nanocomposites for biomedical applications. Recent Advances in Elastomeric Nanocomposites 2011;9: 255–78. 41. El-Fray M, Piegat A, Prowans P. Influence of TiO2 nanoparticles incorporated into elastomeric polyesters on their biocompatibility in vitro and in vivo. Advanced Engineering Materials 2009;11:B200–3. 42. Bal BT, Yılmaz H, Aydın C, Karakoca S, Yılmaz S¸.. In vitro cytotoxicity of maxillofacial silicone elastomers: effect of accelerated aging. Journal of Biomedical Materials Research B: Applied Biomaterials 2009;89:122–6. 43. Ozdemir KG, Yılmaz H, Yılmaz S. In vitro evaluation of cytotoxicity of soft lining materials on L929 cells by MTT assay. Journal of Biomedical Materials Research B: Applied Biomaterials 2009;90:82–6. 44. Srivastava RK, Rahman Q, Kashyap MP, Singh AK, Jain G, Lohani M, et al. Nano-titanium dioxide induces genotoxicity and apoptosis in human lung cancer cell line, A549. Human and Experimental Toxicology 2013;32:153–66. 45. Lu N, Zhu Z, Zhao X, Tao R, Yang X, Gao Z. Nano titanium dioxide photocatalytic protein tyrosine nitration: a potential hazard of TiO2 on skin. Biochemical and Biophysical Research Communications 2008;370:675–80. 46. Schilling K, Bradford B, Castelli D, Dufour E, Nash JF, Pape W, et al. Human safety review of nano titanium dioxide and zinc oxide. Photochemical and Photobiological Sciences 2010;9:495–509.
Available online at www.sciencedirect.com
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Biomechanical properties of nano-TiO2 addition to a medical silicone elastomer: The effect of artificial ageing Linlin Wang a,1, Qi Liu a,1, Dongdong Jing b, Shanyu Zhou a, Longquan Shao a,* a b
Department of Stomatology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China Department of Pharmacy, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
article info
abstract
Article history:
Objectives: The aim of this study was to evaluate the effect of TiO2 nanoparticles on the
Received 13 October 2013
mechanical and anti-ageing properties of a medical silicone elastomer and to assess the
Received in revised form
biocompatibility of this novel combination.
29 December 2013
Methods: TiO2 (P25, Degussa, Germany) nanoparticles were mixed with the silicone elasto-
Accepted 3 January 2014
mer (MDX4-4210, Dow Corning, USA) at 2%, 4%, and 6% (w/w) using silicone fluid as diluent (Q7-9180, Dow Corning, USA). Blank silicone elastomer served as the control material. The physical properties and biocompatibility of the composites were examined. The tensile
Keywords:
strength was tested for 0% and 6% (w/w) before and after artificial ageing. SEM analysis was
Nano-TiO2
performed.
Silicone elastomer
Results: TiO2 nanoparticles improved the tensile strength and Shore A hardness of the
Biomechanical properties
silicone elastomer (P < 0.05). However, a decrease in the elongation at break and tear
Artificial ageing
strength was found for the 6% (w/w) composite (P < 0.05). All the ageing methods had no effect on the tensile strength of the 6% (w/w) composite (P > 0.05), but thermal ageing significantly decreased the tensile strength of the control group (P < 0.05). Cellular viability assays indicated that the composite exhibited biocompatibility. Conclusions: We obtained a promising restorative material which yields favourable physical and anti-ageing properties and is biocompatible in our in vitro cellular studies. # 2014 Elsevier Ltd. All rights reserved.
1.
Introduction
Defects of the maxillofacial area can cause embarrassment for patients. The first choice of treatment is the fitting of a prosthetic following ablative surgery, which can improve the patient’s quality of life.1,2 Silicone elastomer is a promising functional material used for the correction of maxillofacial defects. However, this material does have drawbacks, since natural or outdoor weathering of silicone elastomers can
induce significant changes in its physical and mechanical properties.3 In addition, the conventional disinfection method can accelerate ageing of the facial epitheses.4 Ultimately, this means that the patient must undergo several prosthetic replacements. In this regard, there have been several previous studies on the longevity of facial prostheses, with earlier reports indicating that the average prosthetic lifetime is 6–12 months.5,40 With the continued improvement of clinical care, the demands from both doctors and patients regarding the
* Corresponding author. Tel.: +86 02062787153. E-mail address: [email protected] (L. Shao). 1 These authors contributed equally to this work and should be considered co-first authors. 0300-5712/$ – see front matter # 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jdent.2014.01.002
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performance of silicone elastomers are increasing. Unfortunately, this demand has not been satisfied by the conventional silicone elastomers used in facial epitheses. More recent modifications to silicone elastomers have improved their properties. For example, Liu et al.6 added hollow microspheres into the MDX4-4210 elastomer to decrease density and improve flexibility, and showed that a 5% (v/v) hollow microsphere composite yields a light, soft, flexible, and biocompatible maxillofacial prosthetic material. Han et al.7 found that the incorporation of nano-oxide (Ti, Zn, or Ce) improved the mechanical properties of the A-2186 elastomer (FACTOR II, USA). However, to the best of our knowledge there have been no reports describing improvements to the anti-ageing properties of facial epitheses used in the oral environment. Titanium dioxide (nano-TiO2) nanoparticles have been proposed as a reinforcing material for addition to dental composites. Elsaka et al.8 showed that incorporation of nanoTiO2 nanoparticles into conventional glass-ionomer (GI) improved its mechanical and antibacterial properties. Mixing of zirconia nanoparticles with a primer or an adhesive increased the bond strength of the adhesive system through reinforcement of the adhesive layer and resin tags.9 On the contrary, the addition of TiO2 and SiO2 nanoparticles to poly(methyl methacrylate) acrylic resins adversely impacts the flexural strength, and this effect is directly related to the concentration of nanoparticles.10 Biocompatibility is the ability of a material to function optimally in a particular application, while having minimal impact on the host response. This is a critical feature that distinguishes materials used in biological applications from other high-tech materials.11,12 The biological and toxicological properties of dental materials are the most important criteria on which clinicians base their decisions for facial epitheses selection.13 The MTT assay is a reliable and sensitive method for detecting cell growth and survival.14 While the studies outlined above indicate that silicone elastomer is the preferred material for maxillofacial epitheses, the rapid ageing of facial epitheses in a service environment frequently underlies why such facial epitheses ultimately fail. With this in mind, the aim of the present study was to improve the performance of silicone elastomer properties. Specifically, we used three artificial ageing tests that simulate the oral environment to determine the effect of nanoparticle additives on the tensile strength of elastomers.
2.
Materials and methods
Test samples were obtained by blending 2%, 4%, and 6% (w/w) proportions of TiO2 (P25, Degussa, Germany) nanoparticles and the medical silicone elastomer (MDX4-4210, DOW Corning, USA) in Teflon molds. Thinners (Q7, DOW Corning, USA) were added at a mass ratio of 1:0.8 to the total mass of MDX44210, and TiO2 nanoparticles were weighed using a balance with an accuracy of 0.0001 g (BSA234S, Sartorius, Germany). Subsequently, the TiO2 nanoparticles and curing agent (maintained at a SE:curing agent mass ratio of 10:1) were mixed with the silicone elastomer using a bull magnetic stirrer (HJ-6, Jintan Fuhua Electric Apparatus Co Ltd, China) in a 50-ml glass beaker. Blank silicone elastomer served as the control
group. Air bubbles in the mixture were eliminated by 30 min incubation in a vacuum oven (SHZ-DIII, Nanhe Zhicheng Science and Technology Development Co Ltd, China). Finally, the mixture was allowed to cure for 2 h at room temperature to allow for gas escape. Further curing was then carried out in a high temperature chamber (YLCD-8000P, KELONG, China) at 60 8C for 4 h. The specimens were stored at 23 1 8C for 24 h before testing.
2.1.
Tensile strength and Elongation at break
Thirty-six type 2 dumbbell-shaped specimens were prepared using a silicone elastomer cutter (N = 36, n = 9) (Fig. 1). The tensile strength was measured according to ISO 37:2005 standard on a servo control computerized tensile testing machine (TH-8201S, Tuobo Machinery Co Ltd, China) at a crosshead speed of 500 mm/min. Tensile strength, TS (MPa), was calculated as follows: F TS ¼ Wb where F is the peak force (N), W is the specimen width of narrow parallel portion (mm), and b is the specimen thickness (mm). The elongation at break, Eb (%), was calculated using the following equation: ðL L0 Þ Eb ¼ 100 b L0 where Lb is the test length at break (mm) and L0 is the initial test length (mm).
2.2.
Tear strength
Twenty-four crescent-shaped specimens were prepared using a silicone elastomer cutter (N = 24, n = 6) (Fig. 2). The tear strength was measured based on the ISO 34-1:2004 standard using a servo control computerized tensile testing machine at a crosshead speed of 500 mm/min. Tear strength, TS (KN/m), was calculated according to the following formula: F TS ¼ d where F is the tearing force (N) and d is the specimen thickness (mm).
2.3.
Shore A hardness
Twelve bar-shaped specimens were prepared in a Teflon mold (70 mm 40 mm 6 mm) (N = 12, n = 3). Each sample was
Fig. 1 – Sample size of tensile strength (mm): A = 152 mm, B = 55 mm, C = 25 mm, D = 13 mm, R = 13 mm.
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The values of tensile strength before and after ageing were recorded.
2.5.3.
Stress fatigue
Hertzian contact testing was performed using an experimental system of high-precision biological materials (Electro Force 3510, BOSE, USA). The specimens were submitted to stress fatigue for 66,700, 133,300, 200,000, and 266,700 cycles at room temperature. The load was applied through a stainless steel plate (10 mm 8 mm 7 mm) at a load force of 75 N, and a speed of 72 times/min. The values of tensile strength before and after ageing were tested.
2.6. Fig. 2 – Sample size of tear strength (mm): A I 110 mm, B = 25 W 0.5 mm, C = 68 W 1.5 mm, D = 45 W 0.2 mm, E = 10.5 W 0.05 mm, R1 = 7.5 W 0.5, R2 = 43 W 0.2, R3 = 12.5 W 0.1, R4 = 9 W 0.2.
read at five points. The surfaces of the specimen were cleaned by alcohol before testing. Shore A hardness was measured by a Shore A durometer (LX-A SCLEROMETER, Shangshen, China) based on the ISO 7619-2008 standard. The mean values were recorded.
2.4.
Scanning electron microscopy
The specimens were sputtered with a thin gold layer using a sputter coater (HITACHI, E-1010, Japan). Subsequently, the morphology of the fractured specimens of silicone rubber was observed using a scanning electron microscope (SEM) (HITACHI, S-3700N, Japan).
2.5.
Anti-ageing test
2.5.1.
Thermal ageing
The thermal ageing tests were run in a high temperature chamber (accurate to 1 8C) (YLCD-8000P, KELONG, China). The 6% (w/w) composite and the blank silicone elastomer were subjected to thermal ageing according to the ISO 188:2007 standard. The ageing procedures were conducted at four temperature (50 8C, 100 8C, 150 8C and 200 8C) for 72 h. The sample was suspended for free state to the stainless grids. The distance between each of the two samples was at least 5 mm, and the distance between the sample and the walls of the chamber was at least 70 mm. The values of tensile strength before and after ageing were recorded.
2.5.2.
UV ageing
The UV ageing experiment was performed in the QUVWeathering Tester (QUV/Spray, Q-La, USA). Conditions were 4 h UV irradiation with a power of 0.68 W/m2/nm at 50 3 8C and 4 h water condensate at 50 3 8C. The specimens were submitted to the UV ageing process based on the ASTM D 458701 standard. The 6% (w/w) composites were exposed to UV irradiation by applying a lamp type UVA-340 for 24, 48 or 72 h.
Cytotoxicity testing (MTT assay)
Circular diaphragms (14 mm diameter, 2 mm thickness) of the 6% composite were prepared. All samples were treated using high-pressure steam sterilization. The samples were placed in a medium (RPMI-1640, GIBCO, USA) with 10% foetal bovine serum (16000-044, GIBCO, USA) at 37 8C for 24 h (specific surface area 3 cm2/mL, ISO 10993-5:2009), filtered through 0.22-mm cellulose acetate filters (Millipore; Sigma) prior to being placed in the refrigerator at 4 8C. The specimens were divided into experimental group, negative control group and positive control group. The positive control group was a medium containing 6.4% phenol solution. Cells were seeded in 96-well cell culture plates at 5.0 103 NIH/3T3 mouse fibroblasts cells mL1 per well. The multiwell plates were incubated at 37 8C, 5% CO2 for 24 h. Each group consisted of 5 replicate wells. The culture medium was aspirated from the wells and equal volumes (100 mL per well) of the extracts were added for a further 24 h. In the negative control wells, 100 mL DMEM was added. In positive control wells, 100 mL DMEM with 0.64% phenol solution was added. Then the multiwell plates were incubated at 37 8C, 5% CO2 for 24 h, 48 h or 72 h, after which each well received 20 mL of MTT (5 mg/mL) for 4 h. MTT was then removed and 100 mL DMSO was added to each well, with shaking for 10 min. Subsequently, the absorbance at 570 nm was measured using a UV– visible spectrophotometer (Molecular Devices, USA). Survival rates of the negative controls were set to represent 100%.
2.7.
Statistical analysis
Data of physical and mechanical tests were analyzed using one-way analysis of variance (ANOVA). Two-way classification ANOVA was utilized in analysis of the MTT assay (a = 0.05). Dunnett’s T3 post hoc test was used to determine differences between any of the groups, with a significance level of P < 0.05.
3.
Results
3.1.
Tensile and tear properties
Table 1 demonstrates the mean tensile and tear strength values. Data of tensile strength showed an insignificant increase for the 2% and 4% (w/w) composites (P > 0.05), followed by significantly increased tensile strength at
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journal of dentistry 42 (2014) 475–483
Table 1 – The physical properties of SE incorporated with TiO2 nanoparticles. Group
Tensile strength (MPa)
SE-control SE-2% (w/w) TiO2 SE-4% (w/w) TiO2 SE-6% (w/w) TiO2
2.65 2.80 3.01 3.29
a
(0.09) (0.25) a (0.35)a,b (0.40) b
Elongation at Break (%) 203.23 254.28 192.83 142.15
a
(31.20) (20.14) b (16.46) a (14.63) d
Tear strength (MPa) 10.21 12.58 10.50 6.03
Shore A hardness (MPa)
a
(1.19) (1.91) a (0.77) a (0.530) b
25.07 28.67 30.53 31.97
(1.130) a (0.84) b (1.13) c (1.65) d
The different superscripts mean significant difference (P < 0.05).
concentrations of the 6% (w/w) group (P < 0.05). There was a notably significant increase in elongation at break for the 2% (w/w) composite (P < 0.05). There was no significant difference between the control and 4% composite (P > 0.05). However, the elongation at break significantly decreased in the 6% (w/w) composite (P < 0.05). There was an insignificant trend of increase followed by decline in the tear strength for the 2% and 4% (w/w) composites (P > 0.05). However, the mixing of 6% (w/w) TiO2 nanoparticles with silicone elastomer significantly decreased the tear strength (P < 0.05).
3.2.
Shore A hardness
Shore A hardness significantly increased with increasing concentration of TiO2 nanoparticles incorporated into the silicone elastomer (P < 0.05).
3.3.
3.4.
Ageing
3.4.1.
Thermal ageing
Fig. 4 presents the mean tensile strength values for the 0% and 6% (w/w) groups before and after thermal ageing. The effect of thermal ageing on the tensile strength of 6% (w/w) was not statistically significant (P > 0.05). However, the 0% (w/w) group presented a significant reduction of tensile strength at 200 8C (P < 0.05).
UV ageing
Fig. 5 shows the mean tensile strength values for 0% and 6% (w/w) groups before and after UV ageing. In this test, the 6% (w/w) group yielded higher tensile strength values than the 0% (w/w) group, irrespective of the ageing interval. The 0% and 6% (w/w) groups did not significantly decrease the effects of UV ageing (P > 0.05), although the 0% (w/w) group showed a trend of declined ageing as exposure time increased.
3.4.3.
3.5.
MTT assay
Table 2 shows that the cell survival rate of the 6% (w/w) group was comparable to that of the negative control group (P > 0.05). However, the positive control had a significant reduction in cell survival rate (P < 0.05). Table 3 shows the effect of incubation periods on the cytotoxicity towards NIH3T3 cells. Specifically, increasing the incubation period from 24 to 72 h leads to significant differences in viability (P < 0.05).
4.
Discussion
4.1.
Tensile and tear properties
SEM examination
A representative SEM photomicrograph of the fracture surface of the concentration difference of the TiO2 group revealed that 2% TiO2 nanoparticles were distributed inside the matrix without agglomeration; however, as the concentration of TiO2 nanoparticles increased to 4% and 6% (w/w), the phenomenon of nano-TiO2 agglomeration was apparent in the composite (Fig. 3).
3.4.2.
statistically significant in the control group and the 6% group (P > 0.05).
Stress fatigue
Fig. 6 presents mean tensile strength values for the 0% and 6% (w/w) groups before and after stress fatigue. The effect of accelerated stress cycling on the tensile strength was not
Prosthetics are subjected to tensile stress from every direction when in use, and therefore increasing tensile strength can prolong the useful lifetime of the prosthetic itself.15 Here, we found that a 6% (w/w) concentration of TiO2 nanoparticles mixed with silicone elastomer significantly improved tensile strength compared to the control group. This increase in tensile strength may be due to the dispersal of nanoparticles in the continuous phase of the silicone elastomer, which leads to increased cross-sectional area and force as well as a crosslinked structure formation of the composite material.16 Thus, we provide direct evidence that filling silicon elastomers with TiO2 nanoparticles is associated with strengthening of the material.
Table 2 – Cell viability of groups. Group
OD570 nm
RGR (%)
Level
Experimental group Negative control group Positive control group
0.49 (0.05) a 0.45 (0.11) a 0.09 (0.01) b
104.26 100 19.15
0 0 4
Note: The different superscripts mean significant difference (P < 0.05).
Table 3 – OD values of incubation periods. Incubation periods 24 h 48 h 72 h
N 15 15 15
OD570 nm a
0.30 0.36 b 0.38 b
SE 0.16 0.19 0.22
Different uppercase letters indicate statistical difference (P < 0.05).
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Fig. 3 – SE filled with TiO2 nanoparticles (a, b, c, d, 40T) and (b, d, f, h, 3500T): (a and b) 0% (w/w), (c and d) 2% (w/w), (e and f) 4% (w/w), (g and h) 6% (w/w).
The muscle actions during chewing, talking and laughing cause the remodelling of facial organs such as eyes, mouth and nose. Thus, the ideal facial epitheses should have a certain degree of flexibility, which can not only avoid the damage of facial epitheses, but also give the facial epitheses a more
natural appearance.17 Since the 2% concentrations of nanoTiO2 increase the elongation at break of the silicone elastomer, this may improve the deformation characteristics of facial epitheses. This may be due to the high specific surface area of nano-TiO2, which is likely to reinforce the contact area and the
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Fig. 4 – Tensile strength versus ageing temperature at 50 8C, 100 8C, 150 8C, 200 8C.
extent of binding.18 Studies show that the interaction between the fine particulate filler and matrix can increase the elongation at break of the filling system.19 However, the concentration of nanoparticles in silicone elastomers at 4% and 6% exhibited a certain degree of agglomeration, in which the molecular chains were fixed more firmly around the nanoparticles, weakening the interaction with the silicone elastomer.20 Kulik et al.21 and Chatterjee22 suggested that the high modulus characteristics of nanoparticles can decrease the elastic modulus of the filling material. However, it is imperative that facial epitheses used for the repair of soft tissue defects retain elasticity. The tear strength is also an important indicator of quality for evaluating facial epitheses. Clinically, the ideal shape of facial epitheses is gradually thinner from the middle to the surrounding. The edge is the weakest part of the facial epitheses and is susceptible to damage from external forces.23 Therefore, tear strength is an important factor to ensure longterm and stable reparative effects of facial epitheses. In this study, increases in the tear strength of the silicone elastomer were insignificant in the presence of 2% and 4% (w/w) TiO2 nanoparticles. This may be consistent with previous studies that examined tensile strength. For example, one study reported that nano-sizes of ZnO nanoparticles may result in a wider range of particle distribution attributable to the increased junctions in the composite due to the nano-size effect. The statement means nanoparticles have the nano-size effect, which could improve the mechanical properties of materials.24 However, 6% (w/w) TiO2 nanoparticles weaken the
Fig. 6 – Tensile strength versus stress fatigue frequency for 66,700 times, 133,300 times, 200,000 times, 266,700 times.
tear strength of silicone elastomers compared to the blank silicone elastomer alone. This could be due to the agglomeration of nanoparticles, resulting in poor interfacial bonding, which might force cracks not only along the cutting, but also down into the micro-defects of the nano-TiO2 filler/elastomer matrix. Usually, TiO2 nanoparticles can bond to polysiloxane. Thus, when the amount of nano-TiO2 is increased, there may be an inadequate amount of polysiloxane to link the nanoparticles effectively, which would lead to a decrease in the interfacial bonding in the nano-TiO2 silicone elastomer material.25
4.2.
We found that the Shore A hardness of blank silicon elastomer was lower than that of nano-TiO2 silicone elastomer. This could be due to dispersing of nanoparticles in the silicone elastomer, which increases the crosslink density, thereby leading to increased hardness.26 Another explanation could be that the nanoparticles affect the elastic modulus of the silicone elastomer.27 The modulus of elasticity of silicone elastomer is proportional to the Shore A hardness.28 The Shore A hardness is a measure of the texture of silicone elastomer, with a value between 25 and 35 indicating similarity with soft tissue, according to the defect area.29 In this study, the Shore A hardness of nano-TiO2 silicone elastomer composite was broadly in the range of ideal values, which might meet the hardness requirements of specific anatomical sites, such as maxillary, orbital, nasal and ear tissues. At these sites, the skin is very thin and the bone and cartilage are very close to the surface, creating the need for different texture requirements on facial epitheses compared to other soft tissues.30
4.3.
Fig. 5 – Tensile strength versus UV-ageing time for 24 h, 48 h, 72 h.
Shore A hardness
Ageing
Prosthetic materials must maintain long-term stability in order to function optimally. The effect of ageing can lead to a decline in physical and mechanical properties of the silicone elastomer, shortening the life of facial epitheses, which ultimately leads to replacement. Therefore, anti-ageing properties are important factors for improving facial epitheses stability. The prosthetic material must also be somewhat able to resist external forces, particularly in the context of oral function. Tensile strength is the load that silicone elastomer
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can bear upon stretching prior to fracture, and dictates the maximum capacity to resist tensile failure.30 The study chose tensile strength as an evaluation index for anti-ageing properties, simulating an artificial ageing environment in vitro to test the performance of silicone elastomer. Hatamleh et al.31 showed that silicone elastomer stored in a dark environment for 6 months presented slightly decreased initial performance, but a sharp decline in properties when exposed to the surrounding environment. The common factors that affect the performance of silicone elastomer materials are temperature, light, and mechanical force. Laboratory trials have attempted to recreate an oral environment, although these do have limitations. One clear difference is that these laboratory ageing processes are not carried out in the presence of saliva. Another difference is that many ageing processes do not occur at the same time in the lab test, and so further clinical research is required.32 As is well known, temperature accelerates oxidationreduction reactions within the internal structure of silicone elastomers. The internal facial epitheses may produce deformation and heat during chewing, swallowing and laughing, which can further promote the ageing of the silicone elastomer. Accordingly, laboratory simulations show that thermal ageing for 72 h at 250 8C can simulate 3 years of clinical facial epitheses usage.33 Additionally, facial epitheses can be affected by temperature, chemical substances and mechanical friction. This is why, in the current study, we performed thermal ageing for 72 h at 50, 100, 150, and 200 8C, which is equal to 0.5, 1, 1.5 and 2 years of clinical usage. After thermal ageing, the 6% group of nano-TiO2 silicone elastomers exhibited better anti-heat ageing performance than the other groups, but the tensile strength of the control group significantly decreased as temperature increased. Another group showed that increased temperature will result in hydrolytic scission at siloxane bonds of the main chain, and oxidation decomposition reactions of the side chain of silicone elastomers.34 Nano-TiO2 is a heat-resistant additive and can thus significantly improve the cross-linking reaction temperature of polysiloxane side groups, which in turn improves the heat ageing properties of the silicone elastomer.22 Furthermore, the thermal oxidation stability of the silicone elastomer is inversely proportional to the size of the additive particles.35 Illumination can engender ageing of silicone elastomers, with shorter wavelengths of higher energy (such as UV) producing the greatest destructive effects.3 Currently there are no estimates of the relationship between experimental UV irradiation of prosthetic materials and clinical usage. Because of the lack of such a connection, UV-ageing time intervals were decided by an association of the thermo-cycling process and clinical usage. The thermal ageing process took 86 h to conduct 2000 thermo-cycles, which is equivalent to 2 years of clinical facial epitheses usage.36,37 Considering the above, UV ageing was carried out for 24, 48, and 72 h, which is roughly equivalent to 0.5, 1 and 1.5 years of clinical wear, respectively. Fig. 5 indicates that the 6% (w/w) group composite and the control group had insignificant differences through the UV ageing process. However, the tensile strength of the control group tended to decrease over the course of UV ageing. This suggests that UV ageing time can be increased in order to draw
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more meaningful conclusions in further studies. Shen et al. showed that gamma rays alter the chemical structure and cross-linking density of silicone elastomer composites.38 Similarly, the duration of UV ageing engenders a proportional increase in hydrolytic reactions and accelerates the scission of the main chain in the elastomer.34,22,35 It has been found that nano-TiO2 has a strong ability to resist UV rays. Indeed, the nanoparticles can not only absorb, but also reflect and scatter UV rays due to their refractive index and optical activity.3 Therefore, modification of silicone elastomer material with TiO2 nanoparticles may also improve resistance to environmental UV irradiation. Minami et al.39 showed that 66,700 rounds of cyclic loading correspond to a 0.5-year period of intra-oral conditions. Therefore, in our study we performed 66,700, 133,300, 200,000, and 266,700 rounds in order to simulate 0.5, 1, 1.5 and 2 years of clinical usage. It also reported a bite force of 75 N, which must be the maximum for repetitive loading cycles. The values in Fig. 6 show that the control group and the 6% (w/w) composite group presented a more satisfactory elastic behaviour in tensile strength tests after stress fatigue. Stress fatigue is essentially generated by the combined effects of mechanical force, oxidation, and temperature. When a force acts on the silicone elastomer material, inhomogeneities of the internal structural network can result in the uneven distribution of stress and fracture of local molecular chains. Fong et al.40 showed that when the silicone elastomer was stressed with small amplitude and low frequency forces under conditions of high temperature and oxygen, the valence force of the molecular chain weakened, and the oxidation of silicone elastomer components increased. The oral cavity is a complex microbial environment filled with saliva, which may also generate local pockets of oxidation, and thus decrease the performance of facial epitheses. Stress fatigue has less impact on silicone elastomer prosthetic material than thermal ageing.
4.4.
MTT assay
In 2009, Miroslawa et al.41 studied the in vitro and in vivo cytotoxicity of silicone elastomer with TiO2 nanoparticles. They revealed that silicone elastomer containing TiO2 nanoparticles was non-toxic by a simple propidium iodide (PI) exclusion test using mouse fibroblast 3T3 cell line. Although the type of silicones we used here differs from that used by Miroslawa et al., our findings were essentially consistent with their study. Other researchers have studied the biocompatibility of room-temperature silicone elastomers using the MTT assay,42,43 and concluded that cell survival in the presence of silicone elastomers was affected by incubation periods. In our current study, we found that cell survival increased proportionally with the length of the incubation period. However, we did not demonstrate stability of the nano-TiO2 silicone elastomer in the context of biological reactions. For the special physical and chemical character, nano-TiO2 has been utilized in many fields and brings great progress in people’s life. However a new problem has turned to be the focus of people’s attention, that is, whether nano-TiO2 may bring about potential health risks to the organism. Srivastava et al.44 found that nano-TiO2 induces toxicity mechanism on
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oxidative stress, genotoxicity and apoptosis of human lung cancer cell line A549. Srivastava et al.45 suggested that the skin can be a potential tissue for photo-oxidative injury. Nano-TiO2 displayed prominent photocatalytic activity by stimulating the formation of protein tyrosine nitration, and the optimum condition for the reaction was around physiological pH. However, a review based on the current weight of evidence of all available data shows that the in vitro genotoxic and photogenotoxic profiles of these nano-structured metal oxides are of no consequence to human health.46 Therefore, the biocompatibility of epitheses material still needs further study prior to clinical application. Finally, since nano-TiO2 has photocatalytic activity, this novel composite material may have a potential application as a kind of antibacterial facial epitheses. Further research is certainly warranted to determine whether other properties of this composite make it suitable for long-lasting and antibacterial facial epitheses.
5.
Conclusion
1. A novel maxillofacial silicone elastomer mixed with TiO2 nanoparticles was synthesized. 2. In summary, silicone elastomer filled with 2% (w/w) TiO2 nanoparticles results in a material with improved physical properties for the maxillofacial prostheses. However, the elongation at break and the tear strength of the 6% (w/w) composite were significantly compromised. 3. The TiO2 silicone elastomer composite conferred the ideal values of Shore A hardness. 4. The TiO2 nanoparticles improve the anti-thermal ageing properties of silicone elastomer. 5. Cytotoxicity tests showed that the silicone elastomer filled with nano-TiO2 particles had short-term biocompatibility.
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Acknowledgments This work was supported by National Natural Science Foundation (50973045 and 31070857), Foundation for Highlevel Talents in Higher Education of Guangdong, China (201168), Research Fund for the Doctoral Program of Higher Education of China (20104433120006), and Foundation of President of NanFang Hospital (2013B013).
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