Diatomite filler for resin composites application – A new approach for materials improvement

The aim of this study was to evaluate physical-mechanical properties, degree of conversion, and chemical stability of a nanohybrid composite containing diatomite as filler. Degree of conversion (DC%) of diatomite-containing composite (Zirconfill ® ) was performed using FTIR immediately, and 1, and 7-days post-curing. SEM was conducted to evaluate the surface of the resin after curing and measure particles size. Also, elemental characterization was performed to verify the major components of the composite through EDS. Mechanical characterization using 3-point bending test was performed prior and after thermo-cycling (10000 cycles) (n=10). Knoop microhardness (KHN) was used to characterize mechanical stability after chemical solutions aggression (water, juice, coffee, coke) up to 28 days (n=10/solution). After data normality evaluation using Shapiro-Wilk, One-way ANOVA and Tukey post hoc were conducted to verify differences between groups for DC% and mechanical properties. Split-plot ANOVA was used to compare groups for microhardness characterization (α=0.05). Immediate DC was 60% and significantly increased up to 80% at 7 days (p<0.05). Flexural strength of the diatomite-containing composite was 136.2 (23.7) MPa and significantly decreased to 75.1 (10.2) as a result of thermo-cycling. The flexural modulus was not significantly affected by the thermo-cycling (p>0.05). All the dietary solutions affected the KHN of the composite up to 21 days. For 28 days, the KHN evidenced and stabilization regarding all the solutions. Diatomite-containing composites present good degree of conversion and relevant mechanical properties and demonstrate time-dependent stability against chemical degradation


Introduction
Resin composites are, currently, the most frequent material used in dental restorative process on clinical practice. The mechanical properties and esthetics support their use to anterior and posterior restorations (Ferracane, 2011). However, the composites are still susceptible to fractures, margin degradation and, secondary caries remains as the most common reason to replacement of restorations (Da Rosa Rodolpho et al., 2011;Mjör et al., 2000;Opdam et al., 2010). In order to improve the properties of the composites, many researchers have been developed with different alternatives to modify the material structure.
However, when looking for the filler particles, the main changes were more related to reducing size and modifying shape. Nowadays, most common particles used are barium glass and zirconium with spheric shape and nanometric or nano and micrometric sizes (nanohybrid composites) (Ferracane, 2011). The nanocomposites, which combine nanoparticles and their clusters, were an evident development of dental materials. It seems legitimate to affirm that these materials, due to their excellent polishing capacity, offer good strength and final aesthetics (Silikas et al., 2005). Moreover, it´s known that size, shape and amount of composite inorganic particles influence its surface roughness after polishing (Ferracane, 2011;Stoddard & Johnson, 1991).
In demand for further improvements in mechanical properties and matrix/filler interactions, some porous fillers have been incorporated in dental composites (Wang et al., 2011). In this sense, a diatomite-containing composite is now available.
Diatomite is a porous silicate derived from diatomaceous algae and it presents large surface area, low density, it has been used to increase mechanical properties in polymeric materials, and it is less expensive than others filler materials (Losic et al., 2009).
Given the recent advances in nanotechnology research, it is possible to control diatomite pore size and morphology which can improve photonic, mechanical, absorptive and diffusive properties (Losic et al., 2009). In addition, this porous silica has been recently used as a drug delivery system for therapeutic and regenerative purposes (Maher et al., 2018;Ruggiero et al., 2014;Tamburaci & Tihminlioglu, 2017).
Apart from that, there is evidence that diatomite incorporation results in higher mechanical properties into polymeric materials for different applications (Cacciotti et al., 2019;Liang, 2009;Wang et al., 2011). By this way, the effects of diatomite incorporation in dental composites could be an interesting alternative regarding the monomer interpenetration trough diatomite pores during polymerization and better entanglement between matrix and filler (Wang et al., 2011) which can result in higher degree of conversion and good mechanical properties. However, from the best of our knowledge, there are feel studies regarding chemical and mechanical properties of diatomite-containing dental composites. Thus, the aim of this study is to perform the physicochemical characterization of a novel nanohybrid composite with diatomite filler content Zirconfill ® and its resistance to degradative conditions in vitro.

Experimental design
All the experiments in this study were performed in vitro. In order to characterize Zirconfill ® (BM4, Maringá, PR, BRA), a single nanohybrid resin composite with the presence of diatomite in the composition. Thus, we performed the following analyses: degree of conversion (DC), polymerization shrinkage, surface and filler characterization, flexural strength, and Knoop Microhardness (KHN).

Degree of conversion
The degree of conversion (DC) of the resin was measured using an FTIR Vertex 70 (Bruker Corporation -Billerica, MA -USA). All the measurements were made using absorbance in near-IR spectra with a 4 cm -1 resolution, 32 scans with a range of 4000 to 1000 cm -1 and 10kHz scanner velocity. Resin samples were inserted into a silicon matrix (5x1mm), then transferred to the FTIR device before polymerization to quantify the carbon double bonds peak. After the initial measurement, the samples were photocured with a photocuring unit Valo Cordless (Ultradent Products Inc., South Jordan, UT, USA) 1200 mW/cm 2 for 40 seconds and the absorbances were measured again to verify the variations in the peak of the double bond. The DC was also measured at 24h and 7 days after polymerization. The following formula was used to quantify the DC of the samples and the values were expressed in percentage: DC(%) = [1 -(Rcured/Runcured)].

Surface and filler characterization
Surface and filler characterization were performed using Scanning Electron Microscopy (SEM). Two samples of the diatomite-containing composite were prepared (2x2mm), using a silicon mold, and photocured using Valo cordless (Ultradent) for 40 seconds. The samples were mounted in metallic stubs and sputtered with gold and analyzed in SEM. Besides, one sample Research, Society andDevelopment, v. 11, n. 16, e268111637738, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i16.37738 4 with the same dimensions was mounted in a stub and sputtered with carbon in order to analyze the elements present in the composite structure by EDS. Regarding filler characterization, 2 g of the composite were placed in a vial and immersed in organic solvent (acetone) and centrifuged until complete removal of the matrix preserving only the inorganic part as described before by Fronza et al. 2015(Fronza et al., 2015 and Ruivo et al., 2019(Ruivo et al., 2019.The SEM and EDS analyses were made in microscope Tescan Mira 3 (Tescan Orsay Holding, Kohoutovice, CZ).

Flexural strength and flexural modulus
In order to evaluate the flexural strength, the ISO 4049 (International Organization for Standardization, 2009) protocol was followed. Briefly, 20 specimens 25 x 2 x 2 mm of the composite were prepared using a metallic matrix and photocured with Valo Cordless (Ultradent) for 20 seconds in three different regions of the sample. After photocuring, the samples were immersed in distilled water for 24h at 37 °C. Then, 10 samples were submitted to a 3-point bending test and the other 10 were submitted to 10000 thermal cycles (5°C -55°C) prior to mechanical evaluation. To perform the 3-point bending test, the specimens were put in a universal testing machine (INSTRON, Northwood, MA, USA) with 1mm/min of crosshead speed. After the specimen fracture, flexural strength (MPa) and the flexural modulus were recorded for comparisons between groups with and with no thermal cycles.

Knoop Microhardness (KHN)
Forty discs (4 x 2 mm) of the diatomite-containing resin were prepared using a silicon matrix on a glass plate. The resin was inserted into the matrix, covered with a polyester strip and photocured for 20 seconds using the Valo Cordless photocuring unit (Ultradent) with 1200mW/cm² of irradiance. Then, the discs were embedded in PVC tubes using acrylic resin. After acrylic resin set reaction, the samples were polished with silicon carbide paper (#400, #600, #1200, and #2000), felt disc and abrasive paste.
After polishing procedures, the Knoop microhardness (KHN) of all samples was evaluated in a micro indenter HMV-2 (Shimadzu -Japan). Three measurements were made on each sample using a load/speed ratio of 50g/5s. The global mean of the samples was obtained, and these data recorded as the baseline. Subsequently, the samples were divided into 4 groups (10 discs/group) and immersed in different solutions (water, coffee, coke, orange juice) and left under 37 °C for 28 days. The solutions were changed every day. Every 7 days, the samples were removed from the solutions, cleaned with distilled water and the microhardness measurements retaken for intragroup comparisons.

Statistical analysis
Data homogeneity for all tests was evaluated using Shapiro-Wilk. Degree of Conversion and Flexural Strength were analyzed using One-Way ANOVA. Knoop Microhardness data were compared using split-plot ANOVA. The comparisons between groups were made with Tukey test considering α=0.05. All the analyses were made using software SPSS Statistic 21 (IBM statistics corp., Armonk, NY, USA).
The overall degree of conversion increased as the time passed.
Source: Own Authors. The particles measurements evidenced particles ranging from ~500 nm to up to ~20 µm. It was possible to visualize small particle clusters, and mostly regular particles distribution after matrix removal. The elemental characterization confirmed the siliconzirconium mixed oxide and identified the presence of silicon (~33 wt%), barium (20.9 wt%), and zirconium (8 wt%). Also, about 3 wt% of aluminum was also evidenced in the EDS analysis. Source: Own Authors.

Flexural strength
Data from flexural strength (Table 1) demonstrated the diatomite-containing resin presented high flexural strength and flexural modulus prior to thermo-cycling. Flexural strength significantly decreased (p<0.05), but the modulus was not significantly imparted after 10.000 thermo-cycles. Table 1 -Mean and standard deviation (SD) of flexural strength and flexural modulus for Zirconfill ® before and after 10000 thermo-cycling. Different lower letters in column represent statistical difference (p<0.05).
Source: Own Authors.

Microhardness
KHN values for Zirconfill ® before and after storage in different solutions are exhibited in Table 2. All the solutions affected KHN of Zironfill ® at 7, 14 and 21 days. There were no differences between 21 and 28 days of storage. Also, no differences between solutions were evidenced for the evaluated time-points.

Discussion
Diatomite has been introduced as a biomedical adjuvant for different purposes (Maher et al., 2018;Tamburaci & Tihminlioglu, 2017) due to its high surface area, high porosity, mechanical properties, and thermal stability (Jing et al., 2013;Losic et al., 2009). The incorporation of this material in composites was tested before (Wang et al., 2011), but neither for commercial material nor involving chemical and mechanical properties association. In this study, we bring evidence that commercial composite with diatomite as a filler can reach a high degree of conversion and mechanical properties while has considerable chemical stability.
Each particle of diatomite presents a well-arranged porosity structure (Jing et al., 2013) ranging from few nanometers to micrometers. Besides, it is purposed that the nano-porous organization of diatomite could allow monomer penetration and cross-reaction through the interior of the particles. It is important to note that some limitations in the degree of conversion are associated with the reduction in the polymer network mobility by its surroundings and structure (I. Sideridou et al., 2002). Thus, this complementary polymerization, through the pores of diatomite, could justify our degree of conversion findings. Moreover, that residual polymerization reaching 80% of conversion also contributes to reduce water sorption and solubility that cause hydrolytic degradation (Ferracane, 2006; I. D. Sideridou & Achilias, 2005) and composite discoloration (I. Sideridou et al., 2002). Meanwhile, the long polymerization could affect polymerization ratio and chain relaxation, but those aspects were not investigated in this study.
Apart from that, the degree of conversion is also a determinant factor to the mechanical strength of the composites where the higher the conversion the better the mechanical properties (Ferracane, 2011;I. Sideridou et al., 2002). By this way, the interlocking and the polymerization of the monomers through diatomite pores also increases the mechanical properties.
Considering the high values of initial flexural strength, the high flexural modulus, and the Knoop microhardness, we confirm that diatomite is acting reinforcing the mechanical properties. In this sense, diatomite-containing composite presented a high flexural strength (~136MPa) which allows its recommendation as a material for occlusal surfaces following the International Standards (International Organization for Standardization, 2009). These findings could be related to the fact that diatomite particles deflect cracks and create frictional forces which increases the strength of the material (Wang et al., 2011). In contrast, the significant decrease in the mean value after thermocycling, below 80 MPa, is probably associated with mechanical deformation in the chemical bonds after extreme temperatures variations (Pieniak et al., 2019), which is a common concern for composite restorations. However, more than five specimens presented mechanical strength higher than that, which is also approved according to ISO 4049 (International Organization for Standardization, 2009) requirements. Furthermore, there was no difference in the flexural modulus before and after thermocycling.
The type of filler influences the handling, esthetics, and mechanical properties of dental composites (Pala et al., 2016).
Here, the elemental characterization confirmed the presence of zirconium, barium and silicon as the manufacturer describes as a mixed particles of silicon and zirconium. The content of barium and silicon indicate that barium glass and diatomite are the major components of the filler. Moreover, the filler type and content influence the microhardness of the composites (Hahnel et al., 2010), and the presence of nanohybrid particles of diatomite and zirconia in this composite contributed to the high initial microhardness values. Nevertheless, the new composite with diatomite exhibited some susceptibility to solvent degradation up to 21 days and after that there was a stabilization. This situation is probably occurred due to the plasticizing effect, that separate the molecules in the polymer network and induce reduction in the microhardness (Ferracane, 2006). It is well understood that dental restorations are affected by saliva and chemical beverages in the oral cavity. Besides, pH and temperature variations in that beverages create negative effects to the matrix filler interface in the composite structure (Pala et al., 2016). On the other hand, the microhardness remained above 100 KHN. The high degree of conversion and the mechanical stability created by diatomite interaction with the matrix possibly inhibits a catastrophic decrease in the microhardness induced by different solutions' pH.
Although this is an in vitro study focused on basic characterization of the composite, the outcomes presented here indicate that diatomite is an interesting particle to be used as filler in dental composites, that improves degree of conversion, mechanical properties, and reduces chemical degradability to dietary solutions. On the other hand, surface properties such as color stability, surface roughness and loss of gloss of these composites are still unexplored. Therefore, further studies can be directed to evaluate these properties and compare diatomite-containing composites to the ones containing other types of filler to stablish their differences and predict its clinical success.

Conclusion
The physicochemical characterization of a novel nanohybrid composite Zirconfill ® , with diatomite content, showed that the porous nature of diatomite promoted excellent physical and mechanical properties, high degree of conversion and low susceptibility to dietary solutions degradation conditions in vitro, constituting a promising alternative as filler for nanohybrid dental composites.