Characterization and Crystallinity of Two Bioactive Sealers: Qualitative and Quantitative Analysis

Abstract

Since the crystallinity of hydroxyapatite created by bioactive sealers could affect their solubility percentage, this study aimed to analyze the degree of crystallinity and mineral maturity of hydroxyapatite crystals formed by bioactive (Cerafil and Endosequence) root canal sealers. Set discs of each sealer were submerged, either in deionized water or phosphate buffer solution (PBS). After 30 days, the crystallinity indices, crystal size, and mineral maturity were determined. The data were statistically analyzed using ANOVA and Student’s t tests with significance set at p < 0.05. After immersion in PBS, Endosequence had the most significant value of mineral maturity (1030/1110_Raman) and PO₄/amide I_FTIR ratio. However, the CO₃/PO₄ ratio was reduced by both solutions, particularly by PBS. There was no significant difference between both bioactive sealers. Compared to Endosequence, Cerafil had the highest crystallinity indices for CI_XRD, CI_FTIR, and CI_Raman. AH-26 had a significantly greater crystal size (p < 0.001). There was no significant difference in the crystal size of the Cerafil and Endosequence bioactive sealers (p > 0.05). Overall, Cerafil and Endosequence successfully formed hydroxyapatite crystals when exposed to PBS, with high CIs obtained by Cerafil. The crystallinity and mineral maturity of Cerafil and Endosequence were comparable. On the other hand, AH-26 failed to produce hydroxyapatite.

1. Introduction

To ensure successful endodontic treatment, the root canal sealer used with a gutta-percha core should form an adhesive mass and fill all gaps between the obturating material and radicular dentin to eliminate bacterial microleakage [1,2,3,4,5]. According to Grossman’s recommendation, the sealer should be bactericidal or at least bacteriostatic, biocompatible, provide a fluid-tight seal, and be insoluble when in contact with tissue fluid [1,2,3,4,5]. AH-26 is an epoxy resin-based root canal sealer. It was found to meet most of the requirements of an ideal root canal sealer [6]. It is composed of methenamine, bismuth oxide, silver or titanium, and calcium tungsten [7].

Although epoxy resin-based root canal sealers have a low rate of solubility [8] and a high possibility of dentin adhesion [9], they have a degree of toxicity with no bioactive properties [10]. Calcium silicate-based materials are widely used owing to their biological and bioactive behaviors [11,12].

Bioceramic-based sealers, including Endosequence, are hydraulic calcium silicate materials composed mainly of tri- and dicalcium silicate, calcium phosphate, zirconium oxide, tantalum oxide, and fillers [13,14]. According to the manufacturer’s instructions, Endosequence does not shrink during the setting reaction and has an ability to form hydroxyapatite when exposed to tissue fluid that provides a chemical bond with radicular dentin and improves its sealing ability [15].

It was proven that all calcium silicate-based root canal sealers, including bioceramic sealers, possess a high solubility percentage that exceeds the acceptable limit [6,8,16,17] that could be reduced by their bioactivity [11]. Bioceramic materials are characterized by their bioactive property, which is defined as the bio-mineralization ability of the material to induce the formation of hydroxyapatite-like crystals on their surface when exposed to tissue fluid. This fact is a result of the reaction between the calcium content of the sealer and the phosphate content of the surrounding solution [18]. This can improve the sealing ability of these materials to the root canal system [19]. Furthermore, hydroxyapatite can reinforce the material and improve its mechanical as well as biological properties [20].

During the last few years, there have been much competition between manufacturers to produce the best sealer types with the maximum adherence to the specifications required. Numerous bioceramic brands have been launched on the market, including Cerafil root canal sealers. According to the manufacturer’s brochure, Cerafil is composed of calcium silicate, calcium phosphate, calcium sulfate, calcium oxide, zirconium oxide, bioactive glass, fillers, and accelerator agents [21]. In endodontics, it is of great interest to use sealers with apatite-forming ability. Recently, it was proven that both Endosequence and Cerafil sealers have bioactive properties associated with hydroxyapatite formation that enable them to resist the tendency toward solubility [11]. The formation of hydroxyapatite has attracted much attention because it chemically bonds to the radicular dentin and enhances the sealing of fillings [22].

In a previous study, the influence of bioactivity on the solubility of both Cerafil and Endosequence was evaluated [11]. It proved that both sealers gained weight and resisted solubility, as hydroxyapatite crystals were formed after immersion in phosphate buffer solution (PBS) to a variable degree. The varied degree of crystalline hydroxyapatite for both sealers may depend on their variable crystallinity degree and mineral maturity.

Crystallinity refers to the degree of crystalline mineral structure (crystal lattice) within a solid material and may influence the material’s hardness and density. In turn, this may affect and reduce its solubility [23]. The crystallinity and mineralization of hydroxyapatite may differ from one material to another depending on their composition, particularly their calcium and phosphate contents. Some materials can be prepared using a mixture of amorphous and crystalline structures. The degree of crystallinity, defined as the specified crystalline structure percentage of the material, can determine the degree of crystalline perfection [24].

To elucidate the factors influencing the hydroxyapatite crystallization attained by bioceramic-based root canal sealers, including the crystal size, organic matrix, and mineral contents, as well as the crystallinity index, this study aimed to determine the crystallinity and mineral maturity of hydroxyapatite crystals formed by both bioceramic root canal sealers (Cerafil and Endosequence) versus the standard epoxy resin-based (AH-26) root canal sealer. The null hypothesis is that there is no significant difference in crystallinity and mineral maturity between the tested sealers.

2. Materials and Methods

2.1. Sample Preparation

King Abdulaziz University’s ethics committee approved this study’s procedures (#165-09-23). Two bioceramic-based sealers, Cerafil (Prevest DenPro, Brahmana, India) and BC-Endosequence (Brassler, Boulevard, Savannah, GA, USA), were used in this investigation. The control was an AH-26 resin-based sealer (Dentsply, De Trey, Sirona, Charlotte, NC, USA). For each sealer, twelve samples were fabricated in the advanced research lab at the Faculty of Dentistry, KAU. A 10 mm diameter and 3 mm height polyethylene mold was filled with either a fresh paste of premixed bioceramic sealers or a fresh mix of AH-26 paste that had been mixed based on the manufacturer’s instructions. The molds were packed between glass slabs and incubated at 37 °C and 100% humidity for 7 days for complete setting.

After the setting was completed, six discs from each sealer were immersed in 10 mL of either deionized water or phosphate buffer solution (PBS) for 30 days. Every 3 days, the solution was renewed. According to ISO/FDIS 23317, the tested samples were maintained at a fixed surface area-to-solution volume ratio [25]. The discs were then dried and prepared for analysis.

2.2. Raman Spectroscopy

Using Micro-Raman Spectroscopy (Senterra, Bruken, Berlin, Germany) with a laser driver (Nd-YAG dropped AIY Jernet), a microscope with confocal pinhole, and a CCD detector (Bruker, GmbH, Germany), the surface of each specimen was analyzed. For every sample, three spectra from various sites were acquired. The mineral maturity was determined based on the area ratio of the apatitic v3PO₄³⁻ phosphate/non-apatitic phosphate bands (≈1030/1110 cm⁻¹) [19,26]. The crystallinity index (CI_Raman) was also determined based on an FWHM of the v1PO₄³⁻ peak of ≈960 cm⁻¹ [27].

2.3. Scanning Electron Microscopy (SEM) Analysis

The surface of each specimen was analyzed using SEM (TESCAN VEGA3, Kohoutovice, Czech Republic) to determine the shape of the crystals and particle size before and after 30 days of immersion either in deionized water or PBS.

2.4. Fourier Transform Infrared Spectroscopy (FTIR)

Two discs of each subgroup were milled to fine powder and analyzed using Fourier Transform Infrared Spectroscopy (FTIR-6100, JASCO, Tokyo, Japan). With a resolution of 1 cm⁻¹, the spectra were recorded in the range of 4000 to 400 cm⁻¹. Three scans of each spectrum were performed for data verification.

The mineralization and crystallinity of each material were determined based on the integrated area at 1720–1585, 1200–900, and 890–830 cm⁻¹ corresponding to amide I (collagen matrix), v₁v₃ phosphate (minerals), and v₂ carbonate bands, respectively. Then, the ratios of phosphate/amide I (PO₄³⁻/amide I) and carbonate/phosphate (CO₃²⁻/PO₄³⁻) were calculated [26,28].

The crystallinity index (CI_FTIR), representing the crystallinity degree of hydroxyapatite, was calculated from each spectrum based on the band intensities (at 700–500 cm⁻¹) of split doublet v₄ phosphate bands at ≈600 and 560 cm⁻¹ [28,29,30].

Table 1. Functional groups and parameters of crystallinity index and mineral maturity extracted from FTIR and Raman spectra.

Measured Parameter	Methods	Formula and Functional Group	Band Location (cm−1)	References
Mineral maturity	Raman	Ratio of integrated area of apatitic v3PO43− phosphate/non-apatitic phosphate bands	≈1030/1110	Farlay et al., 2010 [26]
Mineralization minerals/collagen matrix	FTIR	Ratio of integrated area of v1v3PO43−/Amide I	1200–900/1720–1585	Farlay et al., 2010, Figueiredo et al., 2012 and Verdelis et al., 2007 [26,31,32]
Carbonate/phosphate	FTIR	Ratio of integrated area of v2 CO32−/v1v3PO43−	890–830/1200–900	Farlay et al., 2010, Figueiredo et al., 2012 and Verdelis et al., 2007 [26,31,32]
Crystallinity index Raman (CIRaman)	Raman	FHMW of PO43−	≈960	Sa et al., 2017 [27]
Crystallinity Index (CIFTIR)	FTIR	Band intensities (at 700–500 cm−1) of split doublet v4 phosphate bands	≈600 and 560 cm−1	Lebon, 2010 [30]

illustrates the functional groups and parameters of the crystallinity index and mineral maturity extracted from the FTIR and Raman spectra.

2.5. X-ray Diffraction (XRD)

The ground dry powders of each material (either before or after immersion) were evaluated by XRD (Empyrean, Analytical 2010, Whitefield, Bengalore) at a power of 40 kV and 35 mA. The qualitative analysis was performed by identifying the composition phases. For the quantitative analysis, the crystal size (D nm) of the sharpest peak of the crystalline area was calculated based on Equation (1) [33]. However, Equation (2) was used to calculate the crystallinity index (CI_XRD) based on the height of the peaks at an area of 26–35 2θ [27]:

$$\mathrm{Crystal} \mathrm{size} (\mathrm{D} \mathrm{nm})=\frac{k\lambda }{\mathsf{\beta }\cos \mathsf{\theta }}=\frac{(0.9\times 0.15406)}{(\mathrm{FWHM})\cos \mathsf{\theta }}$$

(1)

$$\mathrm{Crystallinity} \mathrm{index}\mathrm{CI}(\mathrm{XRD})=\frac{h(202)+h(300)+h(112)}{h(211)}$$

(2)

2.6. Statistical Analysis

According to a normality test, the Raman data (including mineral maturity and CI_Raman), FTIR data (including PO₄³⁻/amide I and CO₃²⁻/PO₄³⁻ ratios), and XRD data (including crystal size and CI_XRD) were statistically analyzed using ANOVA and post hoc tests. The CI_FTIR was statistically analyzed using a Student’s t test to compare between the different sealers. The tests were performed by SPSS (Version 20, Munich, Germany) with significance set at p < 0.05.

3. Results

3.1. Raman Spectroscopy

Figure 1 illustrates the Raman spectra of all the tested sealers before (black) and after immersion for 30 days either in deionized water (blue) or PBS (red) at 1200–800 cm⁻¹ and presents the parameters of mineral maturity, Raman spectra (area ratio 1030/1110 cm⁻¹), and crystallinity index (CI_Raman = FWHM of 960 cm⁻¹) (Figure 1A–C). The spectra of both bioceramic sealers show that the intensity of the non-apatitic phosphate band at 1110 cm⁻¹ decreased in water with a further decrease in PBS. In the AH-26 spectra, the value of 1110 cm⁻¹ was diminished by water and shifted by PBS (Figure 1C). However, the apatitic phosphate band at ≈1030 cm⁻¹ became very intense in both sealers in PBS (Figure 1A,B). There were changes in its intensity in AH-26 spectra both when immersed in water an in PBS. Furthermore, the band related to hydroxyapatite (≈960 cm⁻¹) was more intense in Endosequence immersed in PBS (Figure 1D). There was no detection of the 960 cm⁻¹ band in the AH-26 spectra.

Regarding the mineral maturity, there was no significant difference between the three sealers either when dry or when immersed in water (Figure 1E) at p > 0.05. However, upon immersion in PBS, Endosequence had the greatest significant value (mean = 9.41 ± 0.95) and AH-26 had the most significant low value (0.37 ± 0.03) at p < 0.001 (Figure 1E).

Regarding the crystallinity index (CI_Raman) in dry conditions, Cerafil had the greatest significant value (24.66 ± 0.49) compared to that of Endosequence (22.14 ± 0.52) and AH-26 (20.01 ± 1.59) at p < 0.001. It was significantly increased when immersed in water, in both Cerafil and Endosequence (31.91 ± 0.4 and 32.72 ± 0.42, respectively), and was further increased by PBS (48.4 ± 1.55 and 47.57 ± 0.53, respectively), with no significant difference between them (p > 0.05) (Figure 1F). PBS did not affect AH-26.

3.2. Scanning Electron Microscopy (SEM)

Cerafil had particles of varying shapes (sphere and spindle shapes) of sizes ranging from 0.14 to 0.41 µm (Figure 2(CR)). The surface of Endosequence showed uniform sphere-like particles of sizes ranging from 0.16 to 42 µm ((Figure 2(ER)). The particle sizes of both bioceramic sealers decreased (from 0.13 to 0.25 µm and 0.08 to 0.11 µm, respectively) after 30 days of being aged in water (Figure 2(CW,EW)). However, after 30 days of immersion in PBS, Cerafil showed a large entanglement of precipitated cauliflower-like hydroxyapatite crystals (Figure 2(CP)). Additionally, Endosequence showed a similar entanglement of smaller fine cauliflower-like crystals (0.06–0.07 and 0.05–0.06 µm, respectively), separated by small spaces and collagen-like structures (Figure 2(EP)).

The surface of AH-26 showed uniform spherical particles ranging from 0.16 to 0.45 µm (Figure 2(AR)), with no changes in their shape and size during water immersion (Figure 2(AW)). However, the aggregation of clusters of globular particles was detected after immersion in PBS (Figure 2(AP)).

3.3. FTIR Spectroscopy

The FTIR spectra of both bioceramic sealers revealed an increase in the intensity of Amide I bands (1720–1550 cm⁻¹) in water with a decrease in PBS (Figure 3(Ca,Ea)). In Cerafil, there were no obvious changes in v₁v₃ phosphate band intensity (1200–900 cm⁻¹) in water and a decrease in PBS (Figure 3(Cb)); however, it was increased by water immersion, with a further increase in PBS in Endosequence (Figure 3(Eb)). The carbonate band (900–800 cm⁻¹) increased during water immersion and decreased in PBS in both bioceramic sealers (Figure 3(Cb,Eb)). In AH-26, the intensity of amide I bands increased in both solutions, while there was a decrease in the mineral bands (phosphate bands) and an increase in the carbonate band (Figure 3(Aa,Ab), respectively).

Accordingly, the PO₄³⁻/amide I ratio was significantly greater in raw Endosequence (24.06 ± 1.3) followed by raw Cerafil (20.66 ± 3.7), while raw AH-26 had the lowest significant value (5.86 ± 0.26) at p < 0.001. This ratio was significantly increased with water immersion and furtherly increased by PBS in both Endosequecne and Cerafil. There was no change in AH-26 in either solution (Figure 4A).

There was no significant difference in the CO₃²⁻/PO₄³⁻ ratio between both raw bioceramic sealers (p > 0.05). However, the CO₃/PO₄ ratio decreased in both solutions, particularly in PBS (Figure 4B).

The represented hydroxyapatite split doublet/v₄ phosphate bands at ≈600 and 560 cm⁻¹ were obvious when the bioceramic was immersed in the solution, particularly in PBS (Figure 3(Cc,Ec)). This was not applicable for AH-26 due to a lack of band height at 590 cm⁻¹ (Figure 3(Ac)). Figure 3D confirms the hydroxyapatite bands for both bioceramic sealers. Accordingly, the crystallinity index (CT_FTIR) represents the crystallinity degree of hydroxyapatite. Although Cerafil had the greater values both in water (5.35 ± 0.51) and PBS (6.93 ± 0.86), there was no significant difference compared with Endosequence (4.15 ± 0.47 and 4.7 ± 0.51, respectively) at p > 0.05 (Figure 4C).

3.4. XRD Results

3.4.1. Qualitative Analysis of XRD

The XRD spectra revealed the phase composition and the percentage (%) of each sealer before and after immersion (Figure 5A,B and Figure 6A). The spectra of raw Cerafil (Figure 5A(a)) showed calcium silicate; wollastonite-2M [CaO₃Si; card # 00-901-1913], alite [Ca₃O₅Si; card # 00-901-6125], cosite [O₂Si, card # 00-900-0805], calcite [CCaO₃; card # 00-900-0966], lime [CaO; card # 00-900-6743], tricalcium phosphate [Ca₃(PO₄)₂; card # 00-151-7238], calcium sulfate anhydrite [CaSO₄; card # 00-500-0040], zirconia oxide [ZrO₂; card # 00-152-2143], and bismite [Bi₂O₃; card #00-901-2546]. After the immersion period, further phases were detected, including portlandite [CaH₂O₂; card # 00-900-6837] in water and hydroxyapatite [Ca₅HO₁₃P₃; card # 00-901-3627] in PBS (Figure 5A(b,c)).

The spectra of raw Endosequence (Figure 5B(a)) detected wollastonite-2M [CaO₃Si; card # 00-901-1913], alite [Ca₃O₅Si; card # 00-901-6125], Dicalcium silicate [Ca₂(SiO₄); calcite [CCaO₃; card # 00-900-0966], tricalcium phosphate [Ca₃(PO₄)₂; card # 00-151-7238], calcium di-aluminum oxide [CaAl₂O₄; card # 00-200-2888], and zirconia oxide [ZrO₂; card # 00-152-2143]. Further phases were detected, including portlandite [CaH₂O₂; card # 00-900-6837] and/or hydroxyapatite [Ca₅HO₁₃P₃; card # 00-901-3627], after immersion in water and PBS, respectively (Figure 5B(b,c)).

All spectra of AH-26 before and after immersion showed similar phase composition with no big difference in their percentages (Figure 6A(a–c)), including bismite [Bi₂O₃; card # 00-901-2546], quartz [O₂Si; card # 00-412-4074], silver [Ag; card # 00-150-9145], calcium tungstate [Ca(WO₄), card # 00-154-16035], and titanium oxide [Ti₂O₃; card # 00-101-0582].

3.4.2. Quantitative Analysis of XRD

Figure 6B illustrates the data of the crystal size of each sealer before and after immersion in different solutions. In the set dry samples, Endosequence exhibited a significantly smaller size (0.47 ± 0.18 nm), while AH-26 exhibited the significantly largest crystal size (0.78 ± 0.12 nm) (p < 0.001). During the immersion period, the crystal size insignificantly decreased for Cerafil, while it increased for Endosequence, with no significant difference between them (p > 0.05).

Cerafil revealed the greatest significant crystalline index (CI_XRD) compared to the other two sealers in all environments (p < 0.001), particularly after 30 days of aging in PBS (Figure 6C).

4. Discussion

When discussing the biological properties of dental materials, there is some terminology to consider. “A material’s capacity to work in a particular application with a suitable host response” is the definition of biocompatibility. The Endosequence bioceramic-based root canal sealer was first introduced in endodontics as a pre-mixed injectable calcium silicate sealer containing tri- and dicalcium silicate, calcium phosphate, calcium hydroxide, and zirconium oxide [34]. Tantalum oxide was also detected by previous studies [13,14]. This was confirmed by the current XRD spectra, as it detected calcium silicate; wollastonite-2M, alite, and dicalcium silicate; and calcite, tricalcium phosphate, calcium hydroxide, and zirconium oxide. In the presence of water, it sets and forms calcium silicate hydrate and calcium hydroxide, resulting in an alkaline medium that enhances antibacterial and biological potential [14]. Furthermore, it is biocompatible and bioactive, as it reacts with the phosphates of the tissue fluids forming hydroxyapatite [13]. Endosequence had a prolonged setting time and a high degree of solubility that jeopardized its sealing ability in [6,17]. Several improvements were attempted by the manufacturer to solve the prolonged setting times and solubility of bioceramics. New versions of bioceramic-based sealers were introduced, including fast-set Endosequence and Cerafil. According to manufacturer’s instructions, Endosequence is aluminum-free; however, calcium di-aluminum oxide was detected by XRD, and previously, the Al element was also detected by EDX [11]. It seems that aluminum was added for the improvement of its delayed setting time, in turn, reducing its solubility property. There is no information on Cerafil’s composition except the manufacturer’s instructions [21], and only one publication explains the effect of bioactivity on reducing its solubility [11]. According to manufacturer’s brochure, Cerafil is composed of calcium silicate, calcium phosphate, zirconium oxide, calcium oxide, glasses, accelerators, filler, and thickening agents [21]. The resulting XRD data of set Cerafil compositions reported a similar composition (calcium silicate, calcite, lime, tricalcium phosphate, calcium sulfate anhydrite, zirconia oxide, and bismite).

In recent years, the term “bioactivity” has been widely used to refer to a property of bioceramic materials, such as the material’s ability to form hydroxyapatite when in contact with simulated tissue fluid, like PBS [19,35]. This hydroxyapatite layer is chemically equivalent to the tooth structure. It can provide the host with interfacial union [23] and improve the material’s physical properties. The main drawback of bioceramic sealers is their solubility [6,16,17], which has been mitigated by increasing their bioactivity [11]. Several publications have determined hydroxyapatite precipitation on Endosequence’s surface after immersion in PBS [34,36,37]. The precipitation was successfully formed due to the reaction of the calcium content of the sealers with the phosphate content of the PBS [38]. Accordingly, PBS was used in this study, since its composition is similar to simulated body fluid [39].

In a previous study, the physical properties of Cerafil in relation to its clinical application, including setting time, solubility, pH, and surface structure, were compared with those of Endosequence as a function of bioactivity after immersion in PBS. Although both sealers successfully formed hydroxyapatite, Endosequence gained more weight than Cerafil. Furthermore, both sealers had different Ca/P ratios, which may be attributed to their variable calcium and phosphate content. It was previously reported in a former study that the Ca/P ratio was decreased after immersion in PBS, with an insignificant difference between Cerafil (2.06 ± 0.43) and Endosequence (2.1 ± 0.04) [11]. This finding may be attributed to the high phosphate content of the surrounding solution (PBS). This result may affect the hardness and crystallization of hydroxyapatite formation. For this reason, this study was focused on the factors affecting the crystallization of both Cerafil and Endosequence. The degree of hardness and density of the material after bioactivity can be evaluated based on several parameters, including the degree of crystallinity and mineral maturity, that affect the perfection of the formed layer. The literature is limited in this area, and further studies are required to evaluate this characteristic concept in root canal sealers. This study is the first publication that focuses on the crystallinity characterization of Cerafil compared with Endosequence after bioactivity in qualitative and quantitative assessments.

Concerning the qualitative evaluation, the chemical composition groups were identified by Raman and FTIR spectroscopy, while the phase composition was identified by XRD analysis. It was revealed that both raw Cerafil and Endosequence have a relatively similar composition with varying percentages. The difference in their composition was detected using their XRD spectra; raw Cerafil contained calcium sulfate anhydrite, and in contrast, the raw Endosequence spectra contained calcium di-aluminum oxide. This finding was supported by previous EDX analysis, whereby Cerafil showed sulfur, while Endosequence showed aluminum [11]. Both sulfur and aluminum are considered during the setting reaction phase to accelerate the hydration reaction of calcium silicate [40]. The setting reaction and material composition can affect the mineral maturity and crystallinity of the full set material. The setting reaction of Cerafil was faster than that of Endosequence [11]; however, Endosequence may undergo incomplete setting before immersion [8]. Furthermore, the crystallinity can be affected by the amount of calcium silicate hydrate (C-S-H). XRD revealed greater C-S-H in Cerafil (26.3%) than in Endosequence (20.2%). Wollastonite (CaO3Si), which was detected in both bioceramic sealers, is a structure rich in calcium chains and can form an outer layer containing CaO/PO, identical to a hydroxyapatite-like material, that can be identified by XRD [23].

It was suggested that the AL₂O₃ addition of Endosequence to CaSi promotes stability in the set material and increases its crystallinity [41], despite its deficient (incomplete) setting reaction before immersion [8,16].

Regarding the quantitative evaluation, mineral maturity can be calculated based on the area ratio of the apatitic/non-apatitic phosphate band of the Raman spectra (1030/1110_Raman cm⁻¹), PO₄/Amide I_FTIR and CO₃²⁻/PO₄³⁻ _FTIR [19,26,28]. The area ratio of 1030/1110 _Raman cm⁻¹ had the greatest significant values for Endosequence, while the lowest significant values were recorded for AH-26. This finding may be attributed to the decrease in the intensity of the non-apatitic band (1110 cm⁻¹) in the bioceramics after immersion (Figure 1A–C). Moreover, an apatitic phosphate band at 1030 cm⁻¹ was present, in the raw Endosequence spectra, which became more intense after immersion in PBS (Figure 1B). However, it was only detected in Cerafil after immersion in PBS and was at a very low intensity in AH-26 (Figure 1C,D). Similarly, Endosequence had a significantly greater value of the PO₄³⁻/Amide I_FTIR ratio, which was increased by immersion either in water or PBS in both Endosequence and Cerafil. However, the CO₃²⁻/PO₄³⁻ _FTIR ratio decreased in both Endosequence and Cerafil after immersion, particularly in PBS. This may be attributed the increase in the phosphate amount by PBS and/or the decrease in the amount of carbonate by the immersion solution. This was confirmed by XRD, as the calcium phosphate percentage was greater in Endosequence (30.6%) than in Cerafil (11.7). After immersion in PBS, the formed hydroxyapatite percentage was greater in Endosequence (32.4.7%) than Cerafil (21.4%), but none was detected in AH-26 (Figure 4). It was found that the increase in the surface carbonated phase would increase the material’s brittleness [42] and wearing property. Calcium carbonate (calcite) is formed during the CaSi hydration process in the presence of water. Although it is essential for obtaining alkaline pH through the activation of portlandite (CaHO) formation, the greatest carbonate phase may lead to the formation of the decalcification of C-S-H with a poor calcium phase [43]. It was confirmed by XRD that the calcite percentage was greater in Endosequence (11.2%) after immersion than in Cerafil (4.9%). So, the carbonate phase may reduce the crystallinity index of Endosequence more than that of Cerafil.

The crystallinity of materials was influenced by their crystal size in [33], and the intensity of the crystalline phase percentage was related to the amorphous phases that can be calculated from the XRD spectra [27].

The crystal size has a critical role in setting time and, in turn, crystal precipitation after the setting reaction. It was suggested that smaller or fine particles can adhere well to each other, forming a crystalline structure [44]. After immersion in PBS, the CI_XRD of Cerafil was significantly greater than that of Endosequence. This may be attributed to their smaller particle size. However, AH-26 had a significantly larger particle size, so it decreased its CI_XRD (Figure 6B,C). This finding is confirmed by SEM images, as the precipitated cauliflower-like hydroxyapatite crystals induced by Cerafil were bigger than those of Endosequence (0.06–0.07 and 0.05–0.06 µm, respectively), while the latter was separated by small spaces and collagen-like structure (Figure 2(EP)). The crystal size and degree of crystallinity are indicative of the Ca/P ratio, which is also influenced by CaCO₃ [45].

The crystallinity of hydroxyapatite can be calculated based on several parameters: from Raman spectra, based on an FWHM of 960 cm⁻¹ (CI_Raman) [27]; from XRD (CI_XRD), based on the highest intensity of the most crystalline phase; and from FTIR, based on split doublet v₄ phosphate bands at ≈600 and 560 cm⁻¹ (CI_FTIR) [28,29,30]. Due to the various parameters, the result may be different. In XRD spectra, the difference in intensity is related to the sharpness of the peaks, which corresponds to the crystalline phase of the sample. Samples with sharp peaks showed a high content of crystalline phase, and the sample with a less sharp peak showed a high content of amorphous phase.

After immersion in PBS, there was no significant difference between the CI_Raman and CI_FTIR of both sealers, while the greatest significant value of CI_XRD was recorded in Cerafil. All parameters for crystallinity indices were dependent on phosphate peaks in all spectra, particularly after hydroxyapatite precipitation. It was detected in both bioceramic sealers, but it was not detected in AH-26. As the crystal size of hydroxyapatite increases, the v₄vPO₄³⁻ _FTIR (603 and 565cm⁻¹) peak increases [46].

All bioceramic-based sealers can produce apatite-like crystal structures when in contact with simulated tissue fluid [6,36]. Each material has a different apatite-forming capability depending on the amount of Ca²⁺ and H⁻ released during the setting reaction [34]. This explains the greater CI_XRD of Cerafil, as the portlandite by-product detected by XRD was greater in Cerafil (7%) than Endosequence (1.2%) (Figure 5).

The alkalinity induced during the hydration reaction may affect the material strength and crystallinity. It was suggested that the presence of more crystalline CaSi is unable to release sufficient calcium, so it influences a alkaline medium that leads to lower compressive strength than that in amorphous CaSi [47]. Previously, it was proved that although Cerafil induced higher pH in water, its pH was significantly lower in PBS than that of Endosequence [11]. This finding may be the reason for the increase in the CI_XRD of Cerafil. The calcium and phosphorus ions released form the bioceramic sealer were the osteogenic ions responsible for the biocompatibility and apatite-forming ability. It was suggested that the silicon ions were considered as a stronger factor for dentin remineralization. It was determined by a previous study that bioceramic sealers released significantly higher calcium ions than epoxy resin. The released ions decreased gradually over 28 days [16]. The released phosphorous and silicon ions were initially greater from bioceramic sealers compared to epoxy resin, and then, gradually decreased over time [8].

5. Limitation of This Study

One of the limitations of this study is the contrast between the laboratory work and the clinical conditions. The disc specimens were tested on a mold, which did not take into consideration the influence of bioactivity in the presence of radicular dentin. More research is needed to determine the impact of the sealer’s bioactivity on sealing ability and tooth fracture resistance.

6. Conclusions

Both Cerafil and Endosequence successfully produced hydroxyapatite crystal-like structures when exposed to the phosphate buffer solution. The mineral maturity of Endosequence was elevated by an increase in its calcium phosphate content, particularly after immersion in PBS, as indicated by PO₄³⁻/Amide I_FTIR, CO₃²⁻/PO₄³⁻ _FTIR, and 1030/1110_Raman cm⁻¹. However, due to the small crystal size of Cerafil after immersion in PBS, it created a greater CI_XRD. The environmental factors, water and PBS, affected the crystallinity and mineral maturity of the sealers. The small particle size of Cerafil and Endosequence promoted the particles’ agglomeration to form hydroxyapatite crystals. Overall, there was no big difference in the crystallinity and mineral maturity of both Cerafil and Endosequence. On the other hand, AH-26 failed to produce hydroxyapatite.