The influence of the particle’s size on the setting reaction of sol-gel derived calcium silicate particles

Introduction: This study aims to analyze the influence of particles size of sol-gel derived calcium silicate particles in the setting reaction of bioactive endodontic cements. Materials and Methods: Sol-gel derived calcium silicate particles were synthesized and sieved to separate the particles in different sizes: CS400, CS200, and CS100. A commercial MTA (Control) was used as control. The particle size and the specific surface area were assessed by laser diffraction and nitrogen adsorption. The cements were prepared with water as the liquid for the reaction. The setting time was conducted according to ISO 6876, and the setting kinetics was analyzed by Fourier transformed infrared spectroscopy (FTIR) at different time points between 120s to 72h. Results: The particle size varied from 9.45μm (CS400 ) to 31.01 (Control). The higher specific surface area valuer reached 15.14g/cm in the CS400. The smallest particle sizes, the higher specific surface area, and the lowest setting time were found for CS400 (p < 0.05). Control presented the highest setting time (p < 0.05). The FTIR analyses showed the differences in materials structure over time, with faster hydration and crystallization for CS400. The setting kinetics was slower for Control even when compared to a sol-gel derived group with similar particle size. Conclusion: The route of synthesis and the particle size influences the setting reaction of calcium silicate-based cements. The reduction of particle size for sol-gel derived calcium silicates lead to the acceleration of the setting reaction of the produced bioactive endodontic cement.


Introduction
Bioactive endodontic cements (BECs) are primarily composed of calcium silicate particles that are responsible for the bioactivity, the sealing ability, and the setting of these materials 1,2 . BECs were first developed for root-end filling procedures, but due to their bioactivity and adequate physicochemical properties, they have been used in different regenerative endodontic procedures such as direct pulp capping 3 , root perforation sealing 4 , apexification 5 , and pulpotomy 6 .
Due to their versatility, BECs have been increasingly used and studied in endodontics. Tailoring cement properties by modifying its setting reaction may be an alternative to increase the clinical applicability of these materials.
Several modifications are made in the material's structure to overcome the long setting time of BECs [7][8][9][10][11][12] . Most of these modifications are related to the addition of new components as catalysts of the setting reaction. Changes in powder: liquid ratio, the addition of additives into the calcium silicate particles, and the addition of catalysts into the water are commonly used strategies.
Controlling the morphological features of calcium silicate particles is another strategy that may be used to modify the setting reaction, promoting its acceleration 13,14 .
Calcium silicate particles are usually produced via the melt-quenched method with melting precursors in high temperatures, leading to dense particles' production 15 . Sol-gel has recently emerged as an alternative route to produce calcium silicate particles for endodontic purposes 12,16 . The formation of silicate bonding at room temperature and the solvent removal lead to the production of highly porous particles with increased surface area 12,17 , enhancing the reactivity of cements with water. It is known that controlling the particle size impacts the surface area available for the reaction in melt-quenched particles 13 , reducing the setting time. This effect may be especially considered for sol-gel derived particles with a high specific area. The optimization of particle size in this cements could contribute to facilitate their clinical application and thus, this study aims to analyze the influence of the size of sol-gel derived calcium silicate particles in the setting reaction of BECs.

Particle synthesis
The calcium silicate particles used in this study were synthesized by the sol-gel route, as previously described 12

Particle characterization
The size and surface area of particles were analyzed by laser diffraction and nitrogen adsorption, respectively. A particle size analyzer (CILAS 1180, France) was used, and the particles were dispersed in isopropyl alcohol (Aldrich Chemical; St Louis, MO, USA) for measurements. A laser was focused on the particle dispersion, and the laser diffraction was measured to characterize the particle size distribution for each group. Nitrogen adsorption measurements were carried out using an Autosorb Quantachrome Nova 1200 (Quantachrome Instruments Corporate Headquarters, USA) instrument. The powder-specific surface area was calculated by the Brunauer-Emmett-Teller (BET) method based on the Nitrogen adsorption isotherm data.

Cement preparation
The cements were produced by mixing CS particles in each particle size with distilled water in an mg:µl proportion of 1.25, as established previously 12 .
The cements were hand-mixed by 30s until all the powder was incorporated into the water leading to a homogenous mixture. The Control group was manipulated according to the manufacturer's instructions. The proportion, in this case, was 1:3, and the mix of cements was made manually as well.

Setting time
The setting time analysis was conducted according to ISO 6876:2012 18 .
The powder and liquid were dispensed and mixed. After 120s from the beginning of cement preparation, samples were produced (n=3) into a mold measuring 4mm diameter x 1mm height. With the aid of a Gilmore needle (100g), indentations were produced sequentially in the surface of the specimen until no visible indentation was seen. The time between the mixture and the absence of indentation was recorded as the setting time of the cements. The mean value between the three analysis was considered as the setting time of cements in minutes.

Setting kinetics
The setting reaction was studied considering the changes in chemical bonding in the cements after the hydration process as previously reported 11 .
Fourier Transformed Infrared Spectroscopy (FTIR) was used for the identification of chemical groups during the reaction. A spectrometer (Vertex 70 -Bruker Optics, Ettlingen, Germany), equipped with an attenuated total reflectance device (Platinum ATR-QL; Bruker Optics) was used, and the prepared cements were placed on the top of the ATR device after 120s of mixing. Measurements were sequentially performed between 400 and 4000cm -1 and started at 120s after cements mixing until 72h after setting. During the time between the analysis, the samples were stored at 37ºC in a 100% humidity environment to avoid sample dehydration. The differences in each sample's chemical profile were used to understand changes in material structure during a long-term setting.

Statistical Analysis
Descriptive analyses were performed for the particle size, the specific surface area, and the FTIR analysis. For the setting time, the normality of data was assessed by the Shapiro-Wilk test, and the groups were compared by One Way ANOVA and Tukey test with 5% significance.

Results
The particle size of sol-gel derived particles and the control group is shown in Table 1. The values ranged between 31.01µm for Control group and 9.45µm for CS400 group (p > 0.05). The experimental cements presented a higher specific surface area, ranging between 15.14 g/cm² and 10.48 g/cm², while the specific surface area for the control group was 4.27 g/cm 2 . Statistically significant higher setting times were observed for Control, where a commercial bioactive endodontic cement was tested. CS200 and CS100 groups presented reduced setting times compared to Control and increased setting time compared to CS400 without a statistically significant difference. CS400 group showed a setting time of 16min and 54s, which are statistically significantly lower than all other groups in the present study (p > 0.05). The FTIR analysis of setting reaction is observed in Figure 1. After 120s of mixing, similar peaks were found for the sol-gel derived cement groups (CS400, cm -1 . These peaks are associated with the presence of OHand are reduced overtime during the setting reaction. A slower reduction on OHintensity was found for Control, CS100, and CS200 groups when compared to CS400. As the reaction takes place, the CO3 -2 ⱱ3 reduced its intensity and a shift for a higher wavelength around 930cm -1 assigned to the formation of Si-Oѵ3 asymmetrical stretching. The A CO3 -2 ⱱ2 peak in the region of 1380cm -1 was observed after 5min of reaction in CS400 group. On CS200, CS100 and Control, this change was found only after 10min, 35min, and 24h, respectively. After 72h, all groups presented Si-O peaks in the region 450-600cm -1 and 900-1015cm -1 and a C-O peak at the region between 1380cm -1 and 1450cm -1, indicating the complete setting reaction.

Discussion
Setting time is still a challenge for the clinical application of BECs 1,7,19 .
Among the strategies to facilitate the application of these cements in daily endodontic procedures, the control of material setting is used in several studies 7,9,12,20 . Tailoring surface particle may be an alternative to adjust the setting of the cement. In this sense, the setting reaction of newly developed calcium silicate properties was tested, and sol-gel derived particles with a high specific surface area were used in different particle sizes and compared to a commercial BEC.
The smaller particle size led to a higher specific surface area and resulted in lower setting times with more rapid modifications in setting reaction.
Smaller particle sizes were known to promote an increase in the specific surface area of particles 21 . The influence of the production route in the morphology of particles is studied 22,23 , and remarkable differences are found between melting technique and sol-gel route derived particles. While the first one is based on the bonding of oxide precursors in high temperatures (1500-2000ºC), the second is based on the formation of silicate network through hydrolysis of alkaloids at low temperatures (20-100ºC). The melting technique leads to the formation of dense structures producing particles with a reduced specific surface area due to the low mesoporosity found for these particles 17 . Under other conditions, the high specific surface area of sol-gel derived particles is related to the gel aging in the particle synthesis 22 . As observed in the present study, higher values for the specific surface area were found for CS400, CS200, and CS100, when compared to the melt-quenched commercial cement used as control. This is explained by the formation of a silicate network during the sol-gel synthesis due to the entrapment of the reaction products (ethanol, nitrates) into the formed gel. During the heat treatment (120-700ºC), they are removed from the structure, leaving a vacancy in the material structure, which becomes a pore after the cooling 24 . These pores are responsible for an irregular surface characteristic in these particles leading to the surface area with higher values (Table 1).
An increased surface area in calcium silicate particles allows the formation of a higher number of hydration sites on the surface of particles. The initial surface dissolution leads to the production of a calcium-silicate-hydrated (CSH) and calcium hydroxide (CaOH)2 14,25,26 as a first step that is essential for the primary setting of cements and further hardening. This reaction results in the formation of silica gel and calcium hydroxide over the surface of the particles. The further steps in the cement hardening involve the precipitation of minerals on the gel layer. As observed in Figure 1, the reduction in the O-H-O and the (CaOH)2 peak was faster for CS400 cements, indicating that the formation of the gel phase and increased reaction rate were more rapid smaller particles used in this study. For the bigger particle sizes, the changes in the FTIR analysis were observed in later times of examination. When considering the CS100 and the Control it is found that despite the closer values for particle size, the setting time is statistically higher for the Control. This result corroborates to the understanding of the role of the surface properties on the initial hydration and the setting of these cements.
After the first step of hydration, several phase transformations take place into the cement structure. The shift in SI-O peak from ~930cm -1 to higher wavelengths, the reduction in the OHpeak (2840 cm -1 to 3700 cm -1 ), and the increase in CO3 2-(1380cm -1 to 1450cm -1 ) indicate the formation of C-H-S and the subsequent crystallization reaction 11,14 . All these modifications are observed in the tested cements and happened in early time points as the particle size reduces in the sol-gel derived materials. In Figure 1  The production route and the particle size are the main reason to explain the differences in morphology of studied particles; however, the commercially available MTA used as the control group presents a different composition. While the CS400, CS200, and CS100 were composed of calcium silicate particles alone, the Control is composed of calcium silicate and tricalcium aluminate 19 .
Tricalcium aluminate is used on the mixture to accelerate the MTA reaction 20,28 .
The hydration of tricalcium aluminate is faster and leads to the formation of a diffusion barrier that impairs the further hydration of calcium silicate particles 15 .
The faster hydration of calcium aluminate is desired in this case for a rapid initial hardening of the materials promoting the initial, which allows this material to be The setting reaction of calcium silicate-based cements may be further studied, and exploiting this reaction may help explain the physicomechanical and biological properties and the cement's behavior in vivo. In the present study, the effect of particle morphology on the setting reaction was shown, and the smaller sol-gel derived particles presented lower setting times. Changing the production route and controlling particle size makes it possible to achieve a faster and effective setting for bioactive endodontic cements.

Conclusions
The route of synthesis and the particle size influences the setting reaction of calcium silicate-based cements. The reduction of particle size for sol-gel derived calcium silicates lead to the acceleration of the setting reaction of the produced bioactive endodontic cement.