Biological and nano-indentation properties of polybenzoxazine-based composites reinforced with zirconia particles as a novel biomaterial

Abstract

Introduction:

The biological and mechanical properties of substances are relevant to their application as biomaterials and there are many efforts to enhance biocompatibility and mechanical properties of bio-medical materials.

Objectives:

In this study, to achieve a low rate of shrinkage during polymerization, good mechanical properties, and excellent biocompatibility, benzoxazine based composites were synthesized.

Methods:

Benzoxazine monomer was synthesized using a solventless method. FTIR and DSC analysis were carried out to determine the appropriate polymerization temperature. The low viscosity of the benzoxazine monomer at 70°C attract us to use in situ polymerization after high speed ball milling of the benzoxazine and it mixture with different weight fractions of zirconia particles. Dispersion and adhesion between the ceramic and polymer components were evaluate by SEM. To evaluate the biological properties and toxicity of the polybenzoxazine-based composite samples reinforced with zirconia particles, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay was conducted. The micromechanical properties of each composite were evaluated by more than 20 nanoindentation tests and 3 nanoscratching tests. Surface topography of scratched regions was investigated using Atomic Force Microscopy. Shrinkage was simulated by Materials Studio software.

Results:

SEM images showed good dispersion and adhesion between the ceramic and polymer components. Biocompatibility assay showed excellent in vitro biocompatibility. Nano-indentation force-displacement curves showed matrix, reinforcement and interphase regions in specimens and excellent homogeneity in mechanical properties. The nanoindentation results showed that the addition of zirconia particles to the polybenzoxazine matrix increased the modulus and hardness of the neat polybenzoxazine; however, by adding more than an optimum level of reinforcement particles, the mechanical properties decreased due to the agglomeration of reinforcement particles and weak interphase that cause inappropriate load transferring between matrix and reinforcement particles. Results of nano-scratching tests showed effects of zirconia particles as reinforcement on the coeffiecient of friction of the synthesized composites. Shrinkage simulation showed a low rate of shrinkage for polybenzoxazine in comparison with other low shrinkage polymers, such as Bis-GMA.

Conclusion:

Polybenzoxazine based composites that reinforced with an optimum amount of zirconia particles (60% wt micro and 10% wt nano-particles) could be used as a novel biomaterial duo to its excellent biocompatibility, good mechanical properties, appropriate viscosity and low rate of polymeization shrinkage.

Keywords

Biological materials polybenzoxazine zirconia particles nano-indentation response biocompatibility low shrinkage materials

1. Introduction

Polymer-based composite samples reinforced with ceramic particles utilized in dental application require appropriate mechanical properties, a low rate of shrinkage during polymerization, and good biocompatibility.

A lot of research have been done to generate new materials with appropriate properties, for instance polybenzoxazine-based aerogels that were introduced in 2012 [1]. An important area in dental materials research is producing new materials with low rate of shrinkage, high mechanical stability and good biocompatibility such as Filtek™ Silorane System that were introduced in 2007 to the market with aim of decreasing polymerization shrinkage and prevent clinical challenges associated with high polymerization shrinkage of conventional dental materials such as secondary caries, enamel micro-cracks, post-operative sensitivity, microleakage, marginal staining and debonding [2].

Polybenzoxazine is a newly-developed thermosetting resin that has the combined properties of thermoset and thermoplastic polymers [3]. The possibility for molecular design flexibility allows its use in a range of applications. It features ease of production due to its low melt viscosity before polymerization that improves dispersion among the components with the polybenzoxazine base [3].

Polybenzoxazine exhibits negligible volumetric shrinkage upon curing because of its ring-opening mechanism of polymerization that obtains exactly the dimension required [4,5]. It does not require a catalyst or curing agent and no by-products form during curing [6]. It also features low water absorption [7], good chemical resistance [8], excellent mechanical performance [9], low coefficient of thermal expansion [4], low dielectric constant [6], and low cost of raw materials [10]. One drawback is the brittleness of polybenzoxazine [7,9,11,12]. Alloying polybenzoxazine with other polymers can reduce this drawback; the use of thermoplastic polymers and nano-particles can toughen polybenzoxazine [13].

Zirconia (ZrO₂) is used in bioengineering for sensors, environmental filters, mechanical components, orthopaedic joints, and dental implants such as crowns and bridges because of its excellent mechanical properties and good bio-compatibility [14,15].

The striking properties of polybenzoxazine and zirconia particles were investigated to determine the biological, nano-indentation and nano-scratching response of the composites and to introduce it as a candidate for dental composites and implants. To the best of our knowledge, there are no prior studies have been published on the preparation of polybenzoxazine/μZrO₂ composites by in situ polymerization and that evaluate the biological properties, nano-indentation, and nano-scratching response of both nano- and micro-composites of polybenzoxazine/zirconia.

2. Materials and methods

2.1. Materials

Bisphenol A (polycarbonate grade, Molar mass: 228.29 g/mol), para-formaldehyde and aniline (Molar mass: 93.13 g/mol) were purchased from Merck. Micro-ZrO₂ was obtained from Sigma Aldrich with an average diameter of 5 μm. Nano-ZrO₂, with an average particle size of 10–15 nm and true density of 5.68 g/cc was obtained from Tecnan.

2.2. Sample preparation

Benzoxazine monomer was synthesized from bisphenol A, aniline, and para-formaldehyde at a molar ratio of $1 : 2 : 4$ using the solventless method as explained by Ishida [16] shown in Fig. 1. The reactants were mixed at 110°C and stirred continuously for 20 min to achieve a light yellow liquid substance which solidified at room temperature and was then ground to powder [17,18].

Fig. 1.

Chemical structure of synthesized benzoxazine monomer.

Benzoxazine monomer and 30%, 40%, 50%, 60%, 70% (w/w) of micro-zirconia particles were mixed to achieve good dispersion with a high speed ball mill at 28 rps for 10 min. The benzoxazine/zirconia samples were then placed in a die 1.6 cm in diameter and 1.5 mm in depth and pressed at 210°C under 10 MPa in pressure for 10 min. The specimens were cured at 220°C for 3 h in an oven [19]. Benzoxazine monomer was also combined with 10, 20, 25, 30 and 40% (w/w) of nano size zirconia particles using the procedure described for micro-zirconia.

2.3. Chemical characterization

Fourier transform infrared spectroscopy (FTIR) was used to detect the chemical bonds in the benzoxazine molecules using a FTIR spectrophotometer (model S 8400; Japan).

2.4. Thermal analysis

Thermal behaviour of the benzoxazine monomer was investigated by conducting differential scanning calorimeter (DSC; model TA-1A). To determine the appropriate temperature at which benzoxazine monomer polymerizes, 15 mg was placed in a pan and heated at the rate of 10°/min from 25 to 300°C under nitrogen atmosphere [20].

2.5. Microstructure characterization

Dispersion of the zirconia particles in the polybenzoxazine matrix was evaluated by scanning electron microscopy (SEM; Tescan HV: 10–20 kv) after sputter coating of all specimens with thin gold film.

Fig. 2.

Interaction between tip of indenter and specimen during nano indentation test (h_max = maximum penetration depth, $h_{c} = contact depth$ , $h_{f} = final depth$ ).

Fig. 3.

Load-displacement curve in nano-indentation process [21].

2.6. Nano-indentation response

Nano-indentation measures the mechanical properties of a small volumes of material such as reduced elastic modulus and hardness by applying a specific load to the surface (typical load: 1 μN to 500 mN) and local deformation (contact depth: 1 nm to 20 μm) [21] as is shown in Fig. 2. The load and displacement are monitored continuously during testing. A typical load-displacement curve is shown in Fig. 3 [22]. The elastic modulus was calculated using the Oliver and Pharr method [23]. Contact stiffness is calculated using following equation: $\begin{matrix} S = \frac{d P}{d h} \end{matrix}$ Where S is the contact stiffness, P is the indentation load, and h is the indentation depth. The initial stage of the unloading curve is calculated using the following equation: $\begin{matrix} S = \frac{2 β}{\sqrt{π}} \times E_{r} \times \sqrt{A_{c}} \end{matrix}$ Where $E_{r}$ is the reduced modulus of indentation contact, $A_{c}$ is the contact area of the indenter, and β is the constant geometry of the indenter. Reduced elastic modulus can be calculated using the following equation: $\begin{matrix} \frac{1}{E_{r}} = \frac{1 - ν^{2}}{E} + \frac{1 - ν_{i}^{2}}{E_{i}} \end{matrix}$ Where E is the elastic modulus of the test sample, $E_{i}$ is the elastic modulus of the diamond indenter tip (1140 GPa), $ν_{i}$ is the Poisson’s ratio of the diamond indenter tip (0.07), and ν is the Poisson’s ratio of the sample. Because the indenter is much stiffer than the specimens, the hardness can be calculated as: $\begin{matrix} H = \frac{P_{\max}}{A_{c}} \end{matrix}$ Where $P_{\max}$ is the maximum load in the load-displacement curve, and $A_{c}$ is the contact area of indenter.

A TriboScope system (Hysirton; USA) with a 60° pyramidal type indenter tip was used. The maximum force applied to specimens was 70 μN that achieved within 5 second and removed again in 5 second without any holding time as shown in Fig. 4. In order to gain reliable results, on each synthesized composite samples more than 25 indents were applied on random locations.

Fig. 4.

Triangular loading-unloading function which is applied in nano-indentation test.

2.7. Surface morphology and nano-scratch analysis

The coefficient of friction is the proportion of lateral force to normal force and contains information about the wear resistance of specimens. The TriboScope system (Hysirton; USA) was used with a 60° pyramidal type indenter tip and atomic force microscopy (NanoScope E; Digital Instruments; USA) was employed to analyze the surface of the specimens. To investigate the scratch properties of the composites, three nano-scratch tests were performed per specimen with a scratch length of 4 μm and scratch time of 30 s. The maximum applied force was 70 μN.

2.8. Biocompatibility

Every materials that synthesized in laboratory for purpose of being a biomaterial should achieve standard level from biocompatibility assays and appropriate host response [1].

2.8.1. MTT-assay

The MTT assay is a quantitative and sensitive biological assay which measures the viability, proliferation and activation of cells based on the mitochondrial dehydrogenase enzymes in living cells. The yellow water-soluble 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) converts into purple formazan product which is insoluble in water. The optical density of the formazan crystals was read using an Elisa plate reader and is directly proportional to the number of living cells [24].

Preparation of culture medium. DMEM (10 g) composed of L-glutamine and 3.7 g sodium bicarbonate was mixed with 900 ml deionized water. For each ml of mixture, 100 μg streptomycin, and 100 units of penicillin were added. After complete dissolution, the pH was regulated by the addition of an adequate amount of HCl (1 M at pH 7.2). The culture medium was sterilized in a 0.22 μ filter under laminar flow and kept at 4°C.

Preparation of phosphate buffer saline without calcium and magnesium. Five capsules of PBS (Sigma, USA) were dissolved in 90 ml of deionized water. After complete dissolution, the volume of the solution was increased to 100 ml. It was sterilized under laminar flow and kept at 4°C for further use.

Cell condition. MG63 (NCBI C555) cells were thawed and moved to a flask containing growth medium (10% FBS, DMEM) and the flask was placed in an incubator at 37°C, 5% CO₂, and 90% relative humidity [25].

Extraction. Extraction was carried out according to ISO 10993-5 to evaluate the toxicity of the specimens and its effect on cell proliferation and division. To each specimen (surface area of 3.5 ± 0.5 cm₂), 1 ml of growth medium and extraction were added. After 1, 7, and 14 days, the viability of cells was analysed. And culture medium without extraction of composites was used as negative control sample [26].

Preparation of MTT solution. MTT powder (50 mg) was dissolved in 10 ml of phosphate buffered saline (PBS) to achieve a solution with a density of 5 mg/ml.

Evaluation of viability of cells. Approximately $1 \times 10^{4}$ cells were placed in 96-well tissue culture plates (Nunc; Denmark) and 100 μl culture medium was added to each well and the plates were incubated for 24 h. After adhesion of the cells, the culture medium was removed and 90 μl of composite and 10 μl of FBS were added to the cells. Each specimen was placed in standard culture medium and the plate was incubated for 24 h. At 3 d, the media was removed and 100 μl of MTT solution (0.5 mg/ml) was added to each cell. The plate was incubated for 4 h, after which the MTT was removed and 100 μl of isopropanol (Sigma; USA) was added to dissolve the black sediment. To improve dissolution of the MTT sediment, the plate was placed in a shaker for 15 min. A multiwall microplate reader (ELISA; STAT FAX 2100; USA) was used at a wave number of 545 nm to evaluate the degree of color exchange. The wells with the greatest number of live cells recorded higher optical densities (OD) than wells with a low number of live cells. The toxicity of the composites were calculated as [21,26]; $\begin{matrix} Toxicity = 1 - \frac{mean OD of sample}{mean OD of control} \end{matrix}$

2.8.2. Cell attachment

To investigate the cellular adherence to specimens, 10,000 cells were placed next to each specimen. After 24 h, the cells were fixed to aid adhesion to the surface of the composites using PBS and 4% gulutaraldehyde. SEM was used to investigate the adhesion of cells to specimens [26].

2.9. Theoretical shrinkage simulation

Materials Studio software was used to simulate shrinkage of the benzoxazine and compare it with shrinkage of other dental polymers such as BisGMA and TEGDMA. The structures of the benzoxazine, BisGMA and TEGDMA monomers were designed as shown in Fig. 5. These structures must be optimized with the minimum level of crystal energy. Optimization was done using a smart algorithm that uses a cascade of the steepest descent, and the Newton–Raphson and quasi-Newton methods.

Fig. 5.

Structures of BISGMA, TEGDMA and benzoxazine monomers (grey, white, red and blue atoms represent carbon, hydrogen, oxygen and nitrogen atoms).

The monomer structure indices highlighted essential parameters such as atomic force field and minimum surface energy. Condensed phase optimized molecular potentials for atomic simulation studies (COMPASS) was employed to minimize the surface energy of the monomers. Next, the connectivity indices of the polymer repeat units were determined. Special consideration was made in the design of the polymer chains, such as breakage and construction of bonds during polymerization. The connectivity indices for the polymer chain were used in correlation with the polymer properties.

3. Results and discussion

3.1. FTIR analysis

The FTIR spectrum of the benzoxazine monomer is shown in Fig. 6. The wave number 947 cm⁻¹ is related to the C-H bond, 1190 cm⁻¹ to the C-N-C bond, 1232 cm⁻¹ to the C-O-C bond, 1327 to the CH₂ bond, and 1497 cm⁻¹ to the benzene ring [17]. The FTIR spectrum shows that benzoxazine monomer was synthesized successfully in the laboratory.

Fig. 6.

FTIR Spectrum of synthesized benzoxazine monomer.

3.2. DSC analysis

The polymerization reaction of benzoxazine monomer is shown in Fig. 7. DSC thermogram of the synthesized benzoxazine monomer is also shown in Fig. 8. The exothermic peak of benzoxazine monomer indicates that it polymerizes at approximately 220°C without any need for catalysts for crosslinking in its structure. Based the DSC results, the specimens were cured at 220°C for ring-opening polymerization and crosslinking.

Fig. 7.

Ring-opening polymerization of benzoxazine monomer [27].

Fig. 8.

DSC thermogram of benzaxazine monomer.

3.3. Scanning electron microscopy

The micro-structure of polybenzoxazine-based composite samples reinforced with 50 and 70% (w/w) of micro-zirconia particles were investigated using SEM. The SEM image from polybenzoxazine-based composite reinforced with 50% (w/w) micro-zirconia particles shows good dispersion of zirconia particles in the matrix of the polybenzoxazine as shown in Fig. 9(a). The SEM image of polybenzoxazine-based composite reinforced with 70% (w/w) micro-zirconia particles shows adhesion of the zirconia particles with polybenzoxazine lines as shown in Fig. 9(b). EDS analysis of polybenzoxazine-based composite reinforced with 70% (w/w) micro-zirconia particles also showed Zr in almost every place of specimen. The low viscosity of the benzoxazine monomer at ∼70°C and in situ polymerization led to adequate wetting of the micro-zirconia particles with the polybenzoxazine matrix.

Fig. 9.

(a) SEM image of polybenzoxazine-based composite reinforced with 50% (w/w) micro-zirconia particles. (b) SEM image of polybenzoxazine-based composite reinforced with 70% (w/w) micro-zirconia particles.

3.4. Nano-indentation response

The nano-indentation force-displacement curves for the reduced modulus and hardness obtained using the Oliver-Pharr method are shown in Figs 10 and 11, respectively and Table 1 shows the samples’ symbols which were investigated by nano-indentation and nano-scratching tests. As can be seen a hysteresis loop occurs during loading-unloading process in nano-indentation tests. The force-displacement curves for 4 micro-composites are shown in Fig. 10(a) and force-displacement curves for 3 nano-composites are shown in Fig. 10(b). Curves show different regions in the composite samples with different hysteresis loops, elastic behaviour of zirconia particles as reinforcements showed by loading-unloading curves with higher slope, the hysteresis loops of polybenzoxazine matrix showed its viscoelastic behaviour by lower curve slope, and the interphase between the ceramic and polymer components exhibits a transition region.

Fig. 10.

(a) Nano-indentation force-displacement curves in micro-composites. (b) Nano-indentation force-displacement curves in nano-composites.

Fig. 11.

(a) Reduced modulus of synthesized composites were obtained from average of 25 applied nano-indent. (b) Hardness of synthesized composites were obtained from average of 25 applied nano-indent.

Table 1

Symbols of specimens in nano-indentation tests

Specimens	Symbol
Polybenzoxazine based composite reinforced with 40% (w/w) micro zirconia	PBA + 40% μZrO₂
Polybenzoxazine based composite reinforced with 50% (w/w) micro zirconia	PBA + 50% μZrO₂
Polybenzoxazine based composite reinforced with 60% (w/w) micro zirconia	PBA + 60% μZrO₂
Polybenzoxazine based composite reinforced with 70% (w/w) micro zirconia	PBA + 70% μZrO₂
Polybenzoxazine based composite reinforced with 10% (w/w) nano zirconia	PBA + 10% nZrO₂
Polybenzoxazine based composite reinforced with 20% (w/w) nano zirconia	PBA + 20% nZrO₂
Polybenzoxazine based composite reinforced with 25% (w/w) nano zirconia	PBA + 25% nZrO₂

The results were the average of approximately 25 indentations applied to every synthesized specimen and indicate that the composites having 10, 20 and 25% (w/w) nano-zirconia particles showed an increased rate of reduced modulus values with an increase in the zirconia content of the composites. Reduced modulus of 5.69 GPa, 6.37 GPa and 7.07 GPa were obtained for polybenzoxazine based composites reinforced with 10, 20 and 25% (w/w) nano-zirconia respectively. The high modulus of the nano-zirconia particles and good dispersion between polymer and ceramic components caused these high recorded modulus. The hardness values of the nano-composites fluctuated with the weight fraction of nano-zirconia increased. An increase in nano-zirconia particles to the polybenzoxazine matrix beyond the 20% weight fraction reduced the hardness values because of the weak interface between the ceramic and polymer components. Hardness of 0.62 GPa, 0.65 GPa and 0.63 GPa were achieved for polybenzoxazine based composites reinforced with 10, 20 and 25% (w/w) nano-zirconia respectively.

Composites with 50, 60 and 70% (w/w) of micro-zirconia particles were predicted to have a greater elastic modulus with an increase in zirconia particles. However, the result of 25 indentations at random places on the specimens shows that the modulus decreased as the weight fraction increased from 60 to 70% of micro-zirconia. The reduced modulus for polybenzoxazine based composites reinforced with 60 and 70% (w/w) micro-zirconia were 8.96 GPa and 7.67 GPa respectively. This occurs because agglomeration of the zirconia particles weakened the mechanical properties of the composite. The hardness of the microcomposites decreased with an increase in micro-zirconia particles above 50% (w/w) of zirconia. This occurs because of the decrease in the strong interface between the ceramic and polymer particles with the addition of reinforcement particles. When compared with conventional dental composites, polybenzoxazine-based composites show higher reduced modulus and hardness. Hardness of 0.9 GPa, 0.87 GPa and 0.85 GPa were obtained for polybenzoxazine based composites reinforced with 50, 60 and 70% (w/w) micro-zirconia particles respectively.

The reduced modulus and hardness values for 4 synthesized micro-composites and 3 synthesized nano-composites which were obtained from average of 25 nano-indentation tests, are shown in Figs 11(a) and 11(b), respectively. Also Table 2 contains quantitative results for the reduced modulus and hardness for these samples.

Table 2

Reduced modulus and hardness for synthesized samples

Specimens	Hardness [GPa]	Reduced modulus [GPa]
PBA + 10% nZrO₂	0.62	5.69
PBA + 20% nZrO₂	0.65	6.37
PBA + 25% nZrO₂	0.63	7.07
PBA + 40% μZrO₂	0.63	7.07
PBA + 50% μZrO₂	0.90	7.48
PBA + 60% μZrO₂	0.87	8.96
PBA + 70% μZrO₂	0.85	7.67

3.5. Surface morphology and nano-scratch analysis

The nano-scratch tests’ results and AFM images from one third of the scratch paths are shown in Fig. 12(a)–(f). The coefficient of friction depends on the objects that are causing friction. The value is usually between 0 and 1 but can be greater than 1. A value of 0 means there is no friction at all between the objects. This is only theoretically. All objects in the real world will have some friction when they touch each other. A value of 1 means the frictional force is equal to the normal force. A coefficient of friction that is more than one just means that friction is stronger than the normal force. See Table 3.

Fig. 12.

(a) Nano-scratch results and AFM images of polybenzoxazine based composite reinforced with 30% (w/w) of micro zirconia particles. (b) Nano-scratch results and AFM images of polybenzoxazine based composite reinforced with 40% (w/w) of micro zirconia particles. (c) Nano-scratch results and AFM images of polybenzoxazine based composite reinforced with 60% (w/w) of micro zirconia particles. (d) Nano-scratch results and AFM images of polybenzoxazine based composite reinforced with 10% (w/w) nano zirconia particles. (e) Nano-scratch results and AFM images of polybenzoxazine based composite reinforced with 20% (w/w) nano zirconia particles. (f) Nano-scratch results and AFM images of polybenzoxazine based composite reinforced with 25% (w/w) nano zirconia particles.

Fig. 12.

(Continued.)

Table 3

Average coefficient of friction values for synthesized composites

Specimens	Coefficient of friction
PBA + 10% nZrO₂	0.98
PBA + 20% nZrO₂	1.09
PBA + 25% nZrO₂	1.17
PBA + 30% μZrO₂	1.01
PBA + 40% μZrO₂	1.02
PBA + 60% μZrO₂	1.03

The average of three tests indicates that an increase in micro- and nano-meter size zirconia content of the polybenzoxazine-based composites increases the coefficient of friction. AFM images of the scratch path show the regions through which the indenter passed during nanoscratching test. Polybenzoxazine with a 25% (w/w) of nano-zirconia has the greatest coefficient of friction because of the high specific area of the nano-zirconia and good interphase between the polymer and ceramic particles. Figure 13 shows the zirconia particles in the scratch path and its effect on the coefficient of friction of polybenzoxazine-based composite reinforced with 70% (w/w) micro meter size zirconia particles.

Fig. 13.

Nano-scratch result of PBA + 70% (w/w) μZrO² and AFM image of scratched region.

3.6. MTT assay

The viability of cells was determined by quantitative calorimetric assay (MTT assay). The results showed excellent biocompatibility for the two types of polybenzoxazine-based composites reinforced with micro-zirconia particles at different weight fractions. To increase the reliability of the outcomes from each value of reinforcement particles, the two samples were evaluated. Table 4 shows the cell viability after 1, 7, and 14 days of exposure, results of samples 1 and 4 that contain polybenzoxazine matrix and 70% (w/w) micro-zirconia were similar in comparison with the control sample also results of samples 2 and 5 that contain polybenzoxazine matrix and 50% (w/w) micro-zirconia indicate excellent biocompatibility for these samples. The composites with polybenzoxazine base and zirconia particles as reinforcements exhibited no toxicity and no harmful effects on cells proliferation. See Figure 14.

Table 4
Quantitative amounts of MTT assay

Days Samples Control

1 2 3 4

1 day 0.926 0.980 0.960 0.973 1.000

7 days 0.969 0.970 0.988 0.985 0.971

14 days 0.939 0.873 0.914 0.883 0.937

Average 0.945 0.941 0.954 0.947 0.969

Viability 97.455 97.077 98.418 97.696 100.000

Days	Samples	Control
1 day	0.926	0.980	0.960	0.973	1.000
7 days	0.969	0.970	0.988	0.985	0.971
14 days	0.939	0.873	0.914	0.883	0.937
Average	0.945	0.941	0.954	0.947	0.969
Viability	97.455	97.077	98.418	97.696	100.000

Fig. 14.

Viability and MTT assay of MG63 cells next to the extraction of micro composites with different weight percentage of micro zirconia particles at various time incubations, sample 1 and 3 are composed of PBA + 70% (w/w) μZrO₂, sample 2 and 4 are composed of PBA + 50% (w/w) μZrO₂.

3.7. Cell adhesion

Figure 15 shows SEM images from surface of the three samples containing polybenzoxazine, polybenzoxazine with 50% (w/w) of μZrO₂, and polybenzoxazine with 70% (w/w) of μZrO₂. All images indicate very good adhesion and division of MG63 cells at the surface of the specimens. It can be seen that the surface of the specimens acts as a good support for MG63 adhesion. These images were taken from cells after 1 month exposure to the specimens.

Fig. 15.

SEM images of cell attachment to specimens’ surface after 30 days ((a) polybenzoxazine + 70% (w/w) μZrO₂, (b) polybenzoxazine + 50% (w/w) μZrO₂, (c) polybenzoxazine).

3.8. Shrinkage simulation

Table 5 shows the results of density and shrinkage for four monomers before and after polymerization simulated using Materials Studio. The results show only negligible shrinkage for benzoxazine monomer after polymerization resulting from breakage and construction of covalent bonds during polymerization that produces only a slight difference in volume before and after polymerization. This low shrinkage, makes it a good candidate in the application as polymer based dental materials.

4. Conclusions

Two types of polybenzoxazine base samples reinforced with different amounts of micro- and nano-meter size zirconia particles were fabricated with the aim of providing a negligible polymerization shrinkage rate, suitable mechanical properties, and good biocompatibility in comparison with conventional dental composites. The following results were drown from this study: 1.

The addition of nano-zirconia with an average particle size of 12 nm in comparison with addition of micro sized zirconia particles, decreased the need for pressure in the polymerization process of polybenzoxazine-based composites.

SEM images showed good dispersion of zirconia particles in the polybenzoxazine matrix because of the low viscosity of benzoxazine monomer at 70°C.

Result of 25 nano-indentations applied to each specimen showed that the reduced modulus and hardness of specimens varies according to the percentage of zirconia content. Although the mechanical properties of neat polybenzoxazine improved with the addition of zirconia particles, after a specific amount, agglomeration of particles and inappropriate load transferring between matrix and reinforcement, decreased the mechanical properties.

Nano-scratch tests analysis indicated that addition of nano-zirconia particles increased the coefficient of friction because of the high surface area and good interaction between the matrix and reinforcement particles.

Biological assays which were taken for first time, showed 97% in vitro biocompatibility and good cell adhesion.

Simulation of shrinkage in Materials Studio software showed low shrinkage during polymerization in comparison with conventional low-shrinkage-rate polymers such as BisGMA and TEGDMA.

After all, the analysis’ results of polybenzoxazine based composites that reinforced with an optimum amount of zirconia particles (60% wt micro and 10% wt nano particles) could be used as a novel biomaterial duo to its excellent biocompatibility, good mechanical properties and low rate of polymeization shrinkage.

Table 5

Results of shrinkage simulation

Monomers	Density of monomer	Density of polymer	Simulated shrinkage	Experimental shrinkage
TEGDMA	1.29	1.175	8.9%	9.8% [28]
BisGMA	1.21	1.170	3.3%	3.7% [28]
Benzoxazine	1.22	1.186	2.8%	2.9% [9]

Conflict of interest

The authors have no conflict of interest to report.

References

Rubenstein

D.A.

Mahadik

S.S.

Leventis

and Yin

, Journal of Biomaterials Science, Polymer Edition 23 (2012), 1171–1184.

ESPE, Filtek™ Silorane, Silorane System Adhesive, 2009.

Chernykh

, Case Western Reserve University, 2009.

Ishida

and Low

H.Y.

, Macromolecules, Vol. 30, 1997, pp. 1099–1106.

Liu

and Gu

, Acta Polymerica Sinica 5 (2000), 019.

Ning

and Ishida

, Journal of Polymer Science Part A: Polymer Chemistry 32 (1994), 1121–1129.

Pilato

, Phenolic Resins: A Century of Progress, Springer, 2010.

Kim

H.D.

and Ishida

, Journal of Applied Polymer Science 79 (2001), 1207–1219. doi:10.1002/1097-4628(20010214)79:7<1207::AID-APP80>3.0.CO;2-3.

Ishida

and Allen

D.J.

, Journal of Polymer Science Part B: Polymer Physics 34 (1996), 1019–1030.

10.

and Jana

, Polymers 6 (2014), 1008–1025.

11.

Takeichi

and Agag

, High Performance Polymers 18 (2006), 777–797.

12.

Takeichi

Kawauchi

and Agag

, Polymer Journal, 40 (2008), 1121–1131.

13.

Kiskan

Ghosh

N.N.

and Yagci

, Polymer International 60 (2011), 167–177.

14.

Wise

D.L.

, Encyclopedic Handbook of Biomaterials and Bioengineering: Vols. 1–2. Applications, CRC Press, 1995.

15.

Volpato

C.Â.M.

Bondioli

D’Áltoé Garbelotto

L.G.

and Fredel

M.C.

, Application of Zirconia in Dentistry: Biological, Mechanical and Optical Considerations, INTECH Open Access Publisher, 2011.

16.

Ishida

and Allen

, Physical and mechanical characterization of near-zero shrinkage polybenzoxazines, Polymer Physics (1996).

17.

Ishida

and Agag

, Handbook of Benzoxazine Resins, Elsevier, 2011.

18.

Rimdusit

Jubsilp

and Tiptipakorn

, Alloys and Composites of Polybenzoxazines: Properties and Applications, Springer Science & Business Media, 2013.

19.

Jin

Hou

and Zeng

, Wear 297 (2013), 1025–1031.

20.

Huang

Carson

S.O.

Silva

Agag

Ishida

and Maia

J.M.

, Polymer 54 (2013), 1880–1886.

21.

Molazemhosseini

Tourani

Naimi-Jamal

and Khavandi

, Polymer Testing 32 (2013), 525–534.

22.

Fischer-Cripps

A.C.

, Nanoindentation, Springer, 2011, pp. 21–37. doi:10.1007/978-1-4419-9872-9_2.

23.

Oliver

W.C.

and Pharr

G.M.

, Journal of Materials Research 7 (1992), 1564–1583. doi:10.1557/JMR.1992.1564.

24.

Mosmann

, Journal of Immunological Methods 65 (1983), 55–63.

25.

Hermersdörfer

, Food/Nahrung 39 (1995), 184–185.

26.

Bonakdar

Emami

S.H.

Shokrgozar

M.A.

Farhadi

Ahmadi

S.A.H.

and Amanzadeh

, Materials Science and Engineering: C 30 (2010), 636–643.

27.

Demir

K.D.

Kiskan

Aydogan

and Yagci

, Reactive and Functional Polymers 73 (2013), 346–359.

28.

Atai

Watts

D.C.

and Atai

, Biomaterials 26 (2005), 5015–5020.

Biological and nano-indentation properties of polybenzoxazine-based composites reinforced with zirconia particles as a novel biomaterial

Abstract

Introduction:

Objectives:

Methods:

Results:

Conclusion:

Keywords

1. Introduction

2. Materials and methods

2.1. Materials

2.2. Sample preparation

2.4. Thermal analysis

2.5. Microstructure characterization

2.8. Biocompatibility

2.8.1. MTT-assay

2.8.2. Cell attachment

2.9. Theoretical shrinkage simulation

3.1. FTIR analysis

Table 4 Quantitative amounts of MTT assay Days Samples Control 1 2 3 4 1 day 0.926 0.980 0.960 0.973 1.000 7 days 0.969 0.970 0.988 0.985 0.971 14 days 0.939 0.873 0.914 0.883 0.937 Average 0.945 0.941 0.954 0.947 0.969 Viability 97.455 97.077 98.418 97.696 100.000

4. Conclusions

Conflict of interest

References

Table 4
Quantitative amounts of MTT assay

Days Samples Control

1 2 3 4

1 day 0.926 0.980 0.960 0.973 1.000

7 days 0.969 0.970 0.988 0.985 0.971

14 days 0.939 0.873 0.914 0.883 0.937

Average 0.945 0.941 0.954 0.947 0.969

Viability 97.455 97.077 98.418 97.696 100.000