Comparative evaluation of three nanofilled resin-based dental composites: Cytotoxicity,surface roughness,and flexural properties

Abstract

The clinical performance of dental composite restoration and its overall success are the result of physical, mechanical, and biological properties of the filling materials. The present study aimed to evaluate surface roughness, flexural properties, and cytotoxicity of two nanohybrids, namely, MI Gracefil and Herculite Ultra, and one nanoceramic dental resin composite DiamondLite. The surface roughness was assessed in two different grinding conditions. Flexural properties were measured by, the three-point bending test and the specimens’ fractured surface was analyzed by scanning electron microscopy. Weibull analysis was performed on the flexural strength data to determine the composites’ reliability. The cytotoxicity test was conducted on MC3T3-E1 cells by the MTS assay. The results showed that grinding conditions had a significant effect on all nanocomposites' surface roughness (p < .05). Herculite Ultra had a significantly higher flexural strength (125.8 MPa) and flexural modulus (9.8 GPa) as compared to other two nanocomposites (p < .05). Further, cytotoxicity results indicated that DiamondLite had the lowest cell viability during the incubation period (p < .05). The current study, therefore, concluded that the evaluated nanocomposites showed satisfactory surface roughness. Herculite Ultra flexural properties and Weibull modulus results make it a reliable choice for direct restorations. The cell viability results also showed that all three nanocomposites had moderate cytotoxicity, making them acceptable for direct application in the mouth.

Keywords

Cytotoxicity flexural properties resin-based dental composite surface roughness Weibull statistics

Introduction

Resin composite materials are commonly used in restorative dentistry as dental filling materials.¹ Composite materials have become popular among patients and clinicians because of merits such as esthetics, ease of usage, and their reasonable cost. Dental resin composite formulations are always in evolution and development to achieve a successful restoration. The main components of dental composites are resin matrix as the organic component, fillers (organic or inorganic), initiators, and accelerators.¹ Resin matrix is a mixture of high viscosity and low viscosity monomers.² Various classification systems have been introduced for resin composites based on their application, filler size or type of polymerization. In terms of filler particle size, resin composites can be divided into four groups: hybrid, microfilled, microhybrid, and nanofilled composites. Recent developments focus on filler particle reinforcements, especially nanofillers, to achieve lower polymerization shrinkage and enhanced physico-mechanical properties and esthetics.^3,4 Nanotechnology application in restorative materials over the past few years has been one of the significant advances in dentistry.⁵ Nano-sized filler are incorporated into the resin matrix to improve the composite’s performance.⁶ The first nanocomposite in restorative dentistry was introduced in 2003 by Mitra et al.⁷ Nanohybrids are one of the main types on nanocomposites containing filler particles ranging from 5 to 100 nm form prepolymerized filler (PPF) and ground glass fillers.⁸ PPF fillers are included in resin composites to improve the resin matrix network, reduce polymerization shrinkage, and prevent cracks.⁹ Another type of nanocomposites newly developed and introduced is a nanoceramic resin composite consisting of PolyCrystalline resin matrix and nanoceramic fillers with an oligomer particulate interface. It is claimed that this resin composite has high flexural properties, biocompatibility, and smooth surface after polishing, resulting in excellent esthetics.¹⁰

Since nanocomposites are widely used in anterior restoration, their surface properties, polishability, and surface roughness are always a concern. High surface roughness affects the esthetics, result in discoloration, susceptibility of the restoration to plaque accumulation, and impact the restoration longevity.^6,11,12 Several studies examined polishability, color stability, and wear resistance of these materials.^6,8,13,14 Besides surface properties other factors such as flexural properties of dental composites, which can be influential on the restoration performance under masticatory loads, are crucial in performance of a restoration.¹⁵ Biocompatibility and cytotoxicity are another important concern because of the elution of cytotoxic substances like unreacted monomers in dental resin composites that can be harmful to tissues around the restoration, thus inducing systematic responses.^16,17 Besides the mentioned factors, other criteria such as polymerization shrinkage, secondary caries, and marginal fractures are other material-related drawbacks of dental resin composites.¹⁸ The clinical performance of dental restoration and its overall success is the result of all criteria mentioned above. Although there have been some researches on nanohybrid and nanoceramic resin composites’ surface properties, there is a lack of information regarding these materials' performance in regard to their cytotoxicity and flexural properties which make the investigation of their mechanical and biological properties necessary.^10,14 Especially, Weibull modulus, which determines the reliability of restoration, has not been previously evaluated in nanocomposites. The novelty of our study was evaluation and comparison of these properties which is a unique and new approach in dental nanocomposites evaluation.

Accordingly, the objective of the present study was to evaluate and compare two nanohybrids MI Gracefil and Herculite Ultra and one nanoceramic composite DiamondLite in terms of surface roughness, flexural strength, flexural modulus, and cytotoxicity.

Materials and methods

Study materials

The resin composites investigated in this study were one nanoceramic composite, DiamondLite (DRM Research Lab., Branford, CT, USA), and two nanohybrid resin composites, namely, MI Gracefil (GC Corp., Tokyo, Japan) and Herculite Ultra (Kerr Corp., Orange, CA, USA). All materials were stored in a cool and dry place until the beginning of the experiment. The resin composites' manufacturers and detailed chemical composition of the resin matrix and filler are shown in Table 1.

Table 1.

Resin composites investigated in this study.

Material	Classification	Resin matrix	Filler type	Manufacturer
DiamondLite	Nanoceramic resin composite	PEX (Phenol-Epoxy-Monomer)	Barium Borosilicate glass (78–84 wt%)	DRM, Branford, USA
MI Gracefil	Nanohybrid resin composite	Bis-MEPP, UDMA	Barium silica glass (82.0 wt%)	GC, Tokyo, Japan
Herculite Ultra	Nanohybrid resin composite	Bis-GMA, TEGDMA	Barium Glass, silica, prepolymerized filler (78 wt%)	Kerr, Orange, CA, USA

Bis-MEPP: bisphenol-A-ethoxylate dimethacrylate; UDMA: urethane dimethacrylate, Bis-GMA: Bisphenol-A-diglycidyl methacrylate, TEGDMA: triethyleneglycol dimethacrylate, wt%, weight percentage.

Surface roughness test

Each resin composite’s surface roughness was determined by sample preparation in a mold with the diameter of 8 mm and thickness of 2 mm. Resin composite was placed into the mold, covered with a glass slab and photopolymerized with a LED curing unit (Delight series; Dentall, Seoul, Korea) at a light intensity of 1,200 mW/cm². Thirty samples for each composite were prepared and divided into three groups based on the grinding condition. The groups were as follows: not ground (non-polished), ground by sandpaper #400 grit (polish #400), and ground by sandpaper #80 grit (polish #80). Surface roughness was evaluated by a profilometer (Surfcom 480A-12; Tokyo Seimitsu Co., Tokyo, Japan) after calibration. Surface roughness (Ra, μm) of each specimen was recorded from five readings with a cut off of 0.8 mm and speed of 0.1 mm/s. The mean of the five measurements was recorded to represent the roughness of each sample.

Flexural strength and flexural modulus measurement

The flexural properties of resin composites were evaluated according to ISO 4049.¹⁹ For the three-point bending test, the samples were prepared in a stainless-steel mold (2 × 2 × 25 mm³). Resin composite paste was compressed into the mold and covered by a glass slab. It was then cured by the LED curing unit at a light intensity of 1,200 mW/cm²; in close contact with the slab at the angle of 90° to the specimen in three overlapping parts; each part was cured for 20 s. After the specimens were removed from the mold, they were kept in distilled water at 37°C for 24 h. They were subjected to the universal testing machine (5566S Instron; Canton, MA, USA) at a crosshead speed of 0.5 mm/min until the fracture. The span between the specimen’s support was 20 mm and the maximum fracture load for each specimen was recorded. Flexural strength was calculated according to the following equation

σ_{f} = \frac{3 F l}{2 b h^{2}}

In this equation, the span between supports is l and the maximum fracture load for each sample is F. Also, b is the specimen’s width and h is the specimen’s height. The flexural modulus for each specimen was calculated from the slope of the load-deflection curve in the linear region.

Scanning electron microscopy observation

The fractured samples were collected after the three-point bending test for scanning electron microscopy observation. Each specimen was coated with gold for 5 min at 10 mA current (IC-50; Shimadzu, Kyoto, Japan). After that, gold coat samples were observed by scanning electron microscopy (SEM; JCM-6000 Plus; JEOL, Tokyo, Japan) at an accelerating voltage of 15 kV.

Cytotoxicity evaluation

The mitochondrial dehydrogenase activity of the MC3T3-E1 cells was assessed by the MTS assay to evaluate the cytotoxicity of each resin composite. For each resin composite, five discs were prepared and placed into a 96 well dish. MC3T3-E1 cells with the density of 2 × 10⁴ cells/ml were then seeded on the surface of each sample. The cells were incubated at 37°C and 5% CO₂ for 2, 4and 6 days. At each incubation time point, 25 μl of the MTS solution (CellTiter 96 Aqueous One Solution; Promega Corp., Madison, WI, USA) was added to each well. After 3 h incubation, the media were mixed and absorbance was measured at 492 nm using a plate reader (Multiskan FC; Thermo Fisher Scientific, Waltham, MA, USA).

Statistical analysis

The data were statistically analyzed by ANOVA, using GraphPad Prism version 8 (GraphPad Software Inc., La Jolla, CA, USA), to determine the significant difference among resin composites for each variable (p < 0.05). Tukey’s post-hoc test was then used to determine the difference and analyze each data set.

Weibull analysis was also performed on the flexural strength data to determine each resin composite’s reliability and failure probability. Cumulative failure probability (P_f) was measured using the Weibull statistical function equation, as follows

P_{f} = 1 - \exp [- {(\frac{σ_{c}}{σ_{0}})}^{m}]

In this equation, m is the Weibull modulus, σ_c is the measured flexural strength and σ₀ is the characteristic strength with the failure probability of 63.2%. The Weibull modulus resulted from the slope of the straight line in the plot of ln [ln1/(1-P_f)] against lnσc.

Results

Surface roughness

The surface roughness results for each resin composite based on the polishing condition are shown in Table 2. Two-way ANOVA analysis showed that the grinding condition significantly influenced the surface roughness of all three resin composites. In fact, it was significantly increased in all composites by grinding with sandpaper #400 and sandpaper #80 grit (p < .05). Also, there was a significant difference between Ra values in polish #400 and polish #80. As shown in Table 2, Tukey’s multiple comparisons indicated that there was no significant difference in the surface roughness of three experimental resin composites in the non-polished and polish #400 groups, regardless of the effect of sandpaper smoothness (p > .05). However, in the polish #80 group MI Gracefil with the Ra value of 4.03 ± (0.32) μm, roughness showed significantly higher results in comparison with those obtained for DiamondLite and Herculite Ultra with 2.89 ± (0.38) μm and 3.38 ± (0.34) μm.

Table 2.

Comparison of the mean (±standard deviation) of the surface roughness of each resin composite group.

	Non-polished	Polish #400	Polish #80
DiamondLite	0.33 ± (0.06)^A,a	0.88 ± (0.04)^B,b	2.89 ± (0.38)^C,c
MI Gracefil	0.49 ± (0.08)^D,a	0.79 ± (0.06)^E,b	4.03 ± (0.32)^F,d
Herculite Ultra	0.48 ± (0.03)^G,a	0.84 ± (0.03)^H,b	3.38 ± (0.34)^I,e

Different uppercase letters indicate significant difference among grinding conditions for each resin composite (within the row), and different lowercase letters represent the significant difference among composite resins in each condition (within the column) (p < .05)

Flexural strength and flexural modulus

Figure 1(a) illustrates the results of the flexural strength test in three experimental resin composites. The mean values of flexural strength for DiamondLite, MI Gracefil, and Herculite Ultra were 118.9 ± $17.4$ MPa, 96.2 ± $16.8$ MPa and 125.8 ± $13.4$ MPa, respectively. One-way ANOVA analysis also indicated that MI Gracefil had significantly lower flexural strength values in comparison to those in the two other groups (p < .05). However, DiamondLite and Herculite Ultra were not significantly different in terms of flexural strength. The mean values of flexural modulus for DiamondLite, MI Gracefil, and Herculite Ultra were 7.7 ± $1.2$ GPa, 7.0 ± $1.1$ GPa and 9.8 ± $0.9$ GPa, respectively. One-way ANOVA analysis of flexural modulus results also indicated that Herculite Ultra had a significantly higher value in comparison to the other two composites (p < .05). There was, however, no significant difference in flexural modulus values between DiamondLite and MI Gracefil (p > .05) (Figure 1(b)).

Figure 1.

Bar graph of a. flexural strength and b. flexural modulus. (*p < .05: statistically significant difference).

The characteristic strength and Weibull parameters for each resin composite are shown in Table 3. Herculite Ultra displayed the highest Weibull modulus among three resin composites. Weibull analysis results also revealed that the Weibull modulus of DiamondLite, MI Gracefil, and Herculite Ultra was 7.8, 6.6, and 10.6, respectively. The characteristic strength with 63.2% reliability of DiamondLite, MI Gracefil, and Herculite Ultra was 127.3 MPa, 100.1 MPa, and 130.6 MPa, respectively.

Table 3.

Flexural properties mean (± standard deviation) and Weibull Statistic values for each resin composite.

	Flexural strength (MPa)	Weibull statistics^a			Flexural modulus (GPa)
	Flexural strength (MPa)	m	σ0 (MPa)	R ²	Flexural modulus (GPa)
DiamondLite	118.9 ± ( $17.4$ )	7.8	127.3	0.88	7.7 ± ( $1.2)$
MI Gracefil	96.2 ± ( $16.8)$	6.6	100.1	0.82	7.0 ± ( $1.1)$
Herculite Ultra	125.8 ± ( $13.4$ )	10.6	130.6	0.84	9.8 ± ( $0.9$ )

^aWeibull Statistics

m: Weibull parameter, σ0: Characteristic strength, R²: Regression coefficient.

SEM observation

The SEM images of resin composite’s fractured area after the three-point bending test are represented in Figure 2. The fractured area of each resin composite had a typical topography. Herculite Ultra showed a more smooth surface and homogenous network when compared to the other two resin composites. Some defects and irregularities with a less homogenous surface could also be seen on the DiamondLite and MI Gracefil specimens’ fractured areas after the three-point bending test.

Figure 2.

SEM micrographs of the fractured surfaces: (a) DiamondLite, (b) MI Gracefil, and (c) Herculite Ultra.

Cytotoxicity evaluation

The results of the MC3T3-E1 cells MTS assay, as cultured in direct contact with the resin composite discs after different incubation periods, are shown in Figure 3. The level of cell proliferation on Herculite Ultra was significantly higher than that in the other two resin composites up to 4 days of the incubation period (p < .05). After 6 days, there was, however, no significant difference between cell proliferation in regard to MI Gracefil and Herculite Ultra (p > .05). Despite this, cell viability on DiamondLite was significantly lower at this incubation time, as compared to the other two resin composites (p < .05). The MTS test findings also indicated that although MI Gracefil showed lower cell viability at the 4 day period, its cell viability was significantly increased after 6 days. The cell viability for DiamondLite remained almost steady during the three incubation periods and it was significantly lower than that of Herculite Ultra during all incubation times.

Figure 3.

Absorbance (cell viability) after cell culture at different incubation periods for each resin composite as determined by the MTS assay. Similar lowercase letters indicate no statistically significant difference at each incubation time among resin composites (p < .05).

Discussion

The growing urge for esthetic dentistry makes resin composites an important dental material among clinicians.²⁰ The physical, mechanical and biological properties of a resin composite could affect its performance in the oral environment. Accordingly, this study aimed to evaluate and compare surface roughness, flexural properties and cytotoxicity of three nanocomposites.

Resin composite’s surface roughness is one of the major factors influencing the esthetic performance, plaque accumulation and biofilm formation of the restoration.¹⁴ In the current study, three different grinding conditions were used to evaluate the nanocomposites’ roughness in various clinical situations. Sandpaper #80 grit particle size is similar to the Carborandum points; also, sandpaper #400 grit particle size is like rubber polishing systems such as Diacomp and Enhance.^21,22 As shown, the non-polished group had the smoothest surface, which was related to the higher amount of the resin matrix, as compared to the filler on the composites' surface.²³ Surface abrasion of resin composites starts with the resin matrix abrasion due to its softer nature, followed by abrasion or dislodgment of the filler particles.²⁴ Filler type, load, size, and distribution are the main factors that are influential on the surface of the resin composite.^13,25 Incorporation of nanohybrids results in filler content increase, more homogenous filler distribution and less interparticle distance in dental resin composites, their fine particles could reduce the probability of surface abrasion.^4,26 Furthermore it is believed that abrasion in nanocomposite does not occur with dislodgment of large filler particle size, instead, it arises by removing part of the nanofillers agglomerates that induce a smoother surface. ⁷ Lower roughness of the Herculite Ultra and MI Gracefil after polish with sandpaper #400 compared to the DiamondLite might be attributed to their nanohybrid fillers ranging from 20 to 400 nm making a smoother surface after abrasion. Also, Bis-GMA/TEGDMA copolymer does not make strong bonding to the filler,²⁷ particularly PPF which does not have chemical bond to the resin matrix, so there is a higher probability that part of the filler agglomerates removed from the surface of the composite and made a smooth surface in Herculite Ultra. It has been suggested that abrasive roughness size should be lower than the resin composite filler particles size to attain an acceptable surface in terms of smoothness.²⁸ Since the particle size of Diamondlite ranged from 0.8 to 2.0 μm, which was larger than that in the other two groups, it clarifies the DiamondLite smoother surface after sandpaper #80 polish.

To evaluate flexural performance in our study, flexural strength and modulus of each resin composite were assessed. Besides, since the flexural strength measurement is not enough by itself for the material performance prediction, the reliability of each resin composite was demonstrated by Weibull modulus (m). The higher Weibull modulus for each resin composite indicated its high reliability and the lower chance of the fracture under stress lower than the measured flexural strength.²⁹ Studies have shown that crack propagation and fracture occur at the restoration resin matrix, thus indicating the importance of the resin matrix strength in dental resin composites.³⁰ Resin matrix content and phase, which make the final polymer network, can impact its flexural properties.^31,32 Comparing the Bis-GMA and Bis-MEPP resin matrix showed that Bis-GMA composites contaning had higher flexural strength, as compared to the Bis-MEPP counterparts.³⁰ Also, Bis-GMA represents higher modulus because of its hydroxyl groups compared to the UDMA based composites having rigid hydrogen bonds due to the imino groups.³³ Moreover, a stable and rigid network made by copolymer of Bis-GMA/TEGDMA with fillers that resulted in resin composites with favorable mechanical properties.^34,35 Mentioned factors alongside with its small filler particle size and higher filler proportion, resulting in high-stress absorbance can explain higher flexural strength and modulus of Herculite Ultra.³⁶ The defects on the fractured surface of DiamondLite and MI Gracefil, as compared to Herculite Ultra, could be related to their lower flexural strength and crack propagation at their resin matrix, as mentioned above.

Resin composites’ cytotoxicity is an essential criterion since they are in direct contact with the tooth and oral environment. The residual monomers which are pertained with their degree of conversion and incomplete polymerization are the main reasons for the cytotoxic effects of dental materials on the cells.^37,38 Although some studies explained that after 24 h polymerization, monomer leach, which is associated with the resin composite cytotoxicity, is decreased and can be ignored,³⁹ the results of our study demonstrated that the cytotoxic effect of resin composites was prolonged even for 6 days. Studies showed that in the Bis-GMA/TEGDMA copolymer, the plasticizing effect of TEGDMA and its high polymerization activity have synergic effect; therefore, this copolymer showed high polymerization rate and degree of conversion.⁴⁰ However, the Bis-MEPP/UDMA based composites showed lower degree of conversion which may be related to the central phenyl ring rigidity in the Bis-MEPP that could confine the UDMA conversion rate.⁴¹ Filler proportion is another criterion; higher filler proportion could result in less monomer dissolution and lower cytotoxicity overtime.⁴² Researches suggested that penetration of liquids into the resin composites over time causes surface erosion and release of unreacted monomers, especially Bis-GMA.¹⁷ These factors explain the results obtained in the current study; although, in the first 4 days, Herculite Ultra showed better cell viability, the situation changed overtime, and MI Gracefil displayed higher cell viability after the 6 day incubation.

It should be mentioned that there were some limitations in our study, such as evaluation of the flexural properties in a short time. The long-term evaluation of flexural properties and Weibull modulus is, therefore, recommended. Another limitation was cytotoxicity evaluation assessed only by the MTS assay; further evaluation by live and dead assays is also recommended for future studies.

Conclusion

The findings of the current study indicated that the evaluated nanocomposites showed satisfactory surface roughness. Herculite Ultra had high flexural properties and Weibull modulus, thus making it a reliable choice for direct restorations. The cell viability results also showed that all three nanocomposites had moderate cytotoxicity, making them acceptable for direct application in the mouth. However, Herculite Ultra had higher cell viability, as compared to others. In terms of the overall performance, it can be concluded that based on the criteria evaluated in the current study, among three nanocomposites, Herculite Ultra showed more reliable results and could be therefore the first choice. Among the other two composites, DiamondLite could be our second choice. However, MI Gracefil did not have satisfactory results when compared to the other two groups.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the.This study was supported by JSPS KAKENHI, grant number 19K10228.

ORCID iDs

Shiva Jafarnia

Alireza Valanezhad

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