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 Table of Contents  
Year : 2022  |  Volume : 19  |  Issue : 1  |  Page : 1

In vitro bioactivity of newly introduced dual-cured resin-modified calcium silicate cement

1 Department of Biomaterials, Faculty of Dentistry, Ain-Shams University, Cairo, Egypt
2 Department of Spectroscopy, Physics Division, National Research Centre, Cairo, Egypt

Date of Submission17-Feb-2021
Date of Acceptance23-Jul-2021
Date of Web Publication28-Jan-2022

Correspondence Address:
Dr. Ahmed Elbanna
Department of Biomaterials, Faculty of Dentistry, Ain-Shams University, Cairo
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/1735-3327.336686

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Background: This study was designed to investigate the in vitro bioactivity of a new dual cured calcium silicate cement (TheraCal PT) compared to its light cured (TheraCal LC) and chemically set (Biodentine) counterparts.
Materials and Methods: The study is an in vitro original research article. Prepared cements discs were immersed in deionized water. Ca2+ release was evaluated using inductively coupled plasma-optical emission spectrometry while pH was assessed using a pH meter after 1, 14, and 28 days. Discs for surface characterization were immersed in phosphate-buffered saline (PBS) and were examined using an environmental scanning electron microscope with energy dispersive X-ray (ESEM/EDX), immediately after setting and at 1, 14, and 28 days intervals after that. Attenuated total reflectance (ATR)/Fourier transform infrared (FTIR) and Raman spectroscopy analyses were performed after setting and after 28 days storage in PBS. Statistical analysis was performed using the two-way repeated measure analysis of variance test followed by Bonferroni test for multiple comparisons (P < 0.05).
Results: Biodentine exhibited the highest mean values for Ca2+ release (792,639,278 ppm) and pH (10.99, 12.7, 11.54) at all time intervals. ESEM/EDX displayed a continuous layer of calcium phosphate formed by Biodentine and TheraCal LC while TheraCal PT developed scarce interrupted precipitates after immersion in PBS. ATR/FTIR and Raman spectroscopy for the formed precipitates confirmed the presence of phosphate and Ca (OH) 2 in Biodentine, TheraCal LC and TheraCal PT.
Conclusion: TheraCal PT exhibited limited in vitro bioactivity which may limit its prognosis in clinical applications for vital pulp therapy. TheraCal LC is considered a potential bioactive calcium silicate cement despite its lower Ca2+ release compared to Biodentine. Highest bioactivity was observed in Biodentine.

Keywords: Calcium silicate, dental cements, dental pulp capping, hydrogen ion concentration, pulpotomy

How to cite this article:
Elbanna A, Atta D, Sherief DI. In vitro bioactivity of newly introduced dual-cured resin-modified calcium silicate cement. Dent Res J 2022;19:1

How to cite this URL:
Elbanna A, Atta D, Sherief DI. In vitro bioactivity of newly introduced dual-cured resin-modified calcium silicate cement. Dent Res J [serial online] 2022 [cited 2022 May 23];19:1. Available from: https://www.drjjournal.net/text.asp?2022/19/1/1/336686

  Introduction Top

Bioactive materials are utilized in pulpal and further endodontic procedures to promote healing and minimize the potential for extraction. Tricalcium and dicalcium silicates, the main phases present in commercial portland cements, tend to exhibit such bioactivity. Their action is attributed to their capability to release Ca2+ and initiate apatite crystals precipitation when exposed to phosphate-containing physiological fluids. Mineral trioxide aggregate (MTA), a portland cement-based material, emerged in the early 1990's as a root-end filling material owing to its hydraulic properties and sealing ability.[1],[2]

The term hydraulic calcium silicate cement (HCSC) is used to address the entire family of MTA-like cements.[1] The powder of HCSCs is formed chiefly of dicalcium and tricalcium silicates whose reaction together yields a sticky colloidal calcium silicate hydrate gel that finally solidifies into a hard structure.[3].HCSCs are frequently used in endodontic techniques including pulp capping, pulpotomy, apexogenesis, apexification, perforation repair, and root-end filling thanks to their biocompatibility as well as their setting and sealing ability in moist bloody fields.[4],[5],[6]

Various HCSCs have since emerged to overcome drawbacks of the original MTA cements such as long setting time, difficult handling, solubility and limited radiopacity.[7] Among such HCSCs is Biodentine™ (Septodont) which was formulated as a dentine replacement material.[8] The physical properties of Biodentine were modified via the alteration of powder composition, inclusion of setting accelerators and softeners together with using a predosed capsule configuration to be mixed in a triturator, making its handling more convenient.[9],[10]

Biodentine hydrates in the same way as MTA where tricalcium silicate, calcium carbonate and zirconium oxide are mixed with water, chloride accelerator and a water-soluble polymer. Its advantages over MTA are easier handling, shorter setting time, and improved physicochemical properties.[11] However, different studies revealed a diversification in its setting time which seemed to be longer than that stated by the manufacturer (12 min).[7]

Accordingly, TheraCal LC by Bisco, a light-cured resin-modified calcium silicate-based material emerged to be used as a pulp capping material and a liner under restorative materials.[12],[13] Low solubility and command set were its main merits over Biodentine. TheraCal LC paste is composed of Type III portland cement, fumed silica, strontium glass, barium sulfate (BaSO4), barium zirconate (BaZrO3), and polyethylene-glycol dimethacrylate monomers.[14] The cement is dispensed through a syringe eliminating the need for mixing procedures. It is placed in a thickness of 1 mm to be light cured for 20 s as per the manufacturer's instructions. The polymerization of TheraCal LC is accompanied with low heat production thus reducing adverse pulpal effects when used in pulp-capping techniques.[15]

Considering the resinous nature of TheraCal LC, the hydration of the calcium silicate phase might be somehow ineffectual compared to Biodentine.[16] The actual effect of this phase on pulpal repair is thus questionable due to inadequate moisture diffusion from the pulp–dentine complex to the cement.[7],[17]

Bisco has recently launched new dual cured resin-modified calcium silicate-based cement (TheraCal PT). Its primary indication is pulpotomy since it can be used in adequate thickness. It can also be utilized as a direct and an indirect pulp capping material and as a base under various substrates.

The aim of this study was to evaluate the in vitro bioactivity of the calcium silicate-based cements with different setting mechanisms, the newly developed TheraCal PT (dual polymerization), TheraCal LC (light polymerization) and Biodentine (chemical setting by hydration). The null hypothesis adopted is that there is no difference in bioactivity between the three cements.

  Materials and Methods Top

The study is an in vitro research article evaluating a newly introduced dual-cured resin modified calcium silicate cement.

The products used in this study, their descriptions, setting mechanisms, compositions, manufacturers, and lot numbers are shown in [Table 1].
Table 1: Products' descriptions, setting mechanisms, compositions, manufacturers and lot. numbers

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Specimens' preparation

Disc-shaped specimens (10 mm diameter ×2 mm thickness) were prepared using a split Teflon mold placed on a clean glass slide covered with a celluloid strip. According to the manufacturer's instruction, Biodentine was mixed after adding five drops of the liquid to the loosened powder in the capsule using an amalgamator (3M Capmix™, 3M ESPE, Germany) for 30 s with a speed of 4300 oscillations per minute. The fresh material paste was packed into the mold, covered with a celluloid strip followed by a glass slide while exerting some pressure using a C-shaped clamp to allow the extrusion of excess material according to ISO standard 9917-1:2007 for water-based cements. The mix was left undisturbed in the mold for 1 h to ensure complete setting.

TheraCal LC and TheraCal PT pastes were injected directly in the mold in two successive increments of 1 mm thickness to ensure proper light curing. Each increment was light cured with an light-emitting diode light curing unit (Elipar S10, 3M ESPE, USA) with irradiance 1200 mW/cm2 for 10 s. Before curing the last increment, the materials were covered with a celluloid strip and a glass slide.

Specimens were removed from the molds, immersed individually in sealed 50 mL sterile CELLSTAR® polypropylene tubes (Greiner Bio One International, GmbH, Germany) containing 5 mL deionized water (AccuGENE, Lonza) and stored in an incubator (Titanox, Italy) at 37°C. The leachates were collected from each tube on the 1st, 14th, and 28th days for Ca2+ release and pH measurements. Specimens were then transferred to new tubes containing fresh deionized water.[18]

Concerning surface characterization, (environmental scanning electron microscope with energy dispersive X-ray [ESEM/EDX], Raman micro-spectroscopy, Fourier transform infrared [FTIR] spectroscopy) similar disc-shaped specimens were prepared from each cement for each characterization method. Regarding ESEM/EDX, specimens were examined immediately after setting then after 1, 14, and 28 days of storage in phosphate buffered saline (PBS). As for Raman and FTIR, specimens were analyzed immediately after setting and after 28 days of storage (n = 3).

In vitro bioactivity evaluation

Leachate analysis

pH evaluation

pH of the leachate was evaluated for each material after each storage period via a pH meter (B 712 LAQUA twin compact pH meter, Horiba Scientific, Japan) that was initially standardized by buffered solutions (pH 7) and recalibrated before testing each new specimen. After specimen removal and shaking the storage tube for 5 s, 1 mL from each leachate was aspirated using a micropipette (TopPette, Dragon Laboratory Instruments, China) and was placed in the pH meter measuring lens. For each sample, the pH was measured twice to calculate a mean value.

Calcium release evaluation

Ca2+ release of the tested materials at the predetermined time intervals (n = 7) was measured using inductively coupled plasma-optical emission spectrometry.[19] It was performed through Agilent 5100 Synchronous Vertical Dual View with Agilent Vapor Generation Accessory. Before analysis, the leachates were filtered and acidified using a 10% HNO3 solution. HNO3 with 5, 10, and 15 ppm Ca was used as a series concentration for calibration before measuring the released calcium. The amount of Ca2+ was measured in ppm.

Surface characterization of the cement discs

Environmental scanning electron microscope with energy dispersive X-ray analysis

The surfaces of the specimens were examined without any modifications using an ESEM (TESCAN VEGA3 Czech republic) with attached EDX[20] from Brucker, immediately after setting and after 1, 14, and 28 days of storage in PBS. The discs were examined at different magnifications and elemental analysis was performed at different points on the cements' discs. After each examination, the discs were transferred to fresh 5 mL PBS and stored in the incubator till the next examination.

Attenuated total reflectance/Fourier transform infrared spectroscopy

Infrared (IR) spectra were recorded on a Vertex 70 FTIR (Bruker-Optics, Germany) equipped with attenuated total reflectance (ATR) accessory and a deuterated triglycine sulfate detector. A resolution of 2 cm−1 with a range from 4000cm-1 to 600 cm-1 was utilized. The diameter of ATR accessory was 2 mm diameter and the IR penetration power was about 2 μm. Spectra were accumulated for at least 10 times to avoid any fluctuations in the detection system.

Raman spectroscopy and mapping

The surfaces of prepared samples were first imaged using a bright field microscope showing a nonhomogenized white layer. Such layer was further characterized using Raman spectroscopy. Raman single point measurements and imaging were performed using Alpha 300R/AS, Witec, Germany. High-resolution imaging was achieved through applying the smallest confocal volume. A ×100 objective lens with high numerical aperture was utilized for both single point and surface mapping. The 532 nm laser was used, its power density had been optimized to 70 mW/mm2 by testing the exposed area with different laser powers using an optical microscope. The laser power was doubled in the case of mapping to reduce the exposure time at every point. The resolving grating was 600 g/mm with Blazing = 500 nm. Single point spectra were collected for 900 s for every sample. The mapped surface area was 25 by 25μ with 150 pins, and exposure time was 1 s for every pin. The band at 1040 cm−1 attributed to phosphate (PO4)−3 was used as a specific peak to obtain Raman mapping.

Statistical analysis

IBM SPSS statistics data editor version 24 (IBM Corp, Armonk, New York, USA) was used. For both pH and calcium ions release test, data were checked for normality using the Kolmogorov–Smirnov test and Shapiro test, and data were found to be normally distributed. Two-way repeated measure analysis of variance test was carried out followed by Bonferroni post hoc test for multiple comparisons. Sample size was confirmed with statistical power analysis for each test with observed power more than 0.96. In the tables, different letters represent statistically significant differences (P < 0.05) in the same row (capital letters) or in the same column (lower case letters).

  Results Top

Leachate analysis

pH results

All materials showed an increasing pattern in pH mean values till day 14 followed by an insignificant decrease on day 28, as shown in [Figure 1]. Biodentine leachate showed significantly higher pH values compared to those of TheraCal LC and TheraCal PT at all time intervals (P < 0.05). TheraCal LC leachate exhibited significantly higher pH compared to TheraCal PT on the 1st day only.
Figure 1: Line chart with table representing pH results of Biodentine, TheraCal LC, and TheraCal PT.

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Calcium ion release

[Figure 2] illustrates Ca2+ release data. Biodentine showed a descending pattern in Ca2+ release with significantly lower mean values after 28 days. Despite this decreasing pattern, Bonferroni post hoc test showed that the mean values for Biodentine were significantly higher at all time intervals compared to TheraCal LC and PT. TheraCal LC displayed a decreasing pattern in Ca2+ release for 14 days followed by a significant drop after 28 days. TheraCal PT revealed limited Ca2+ release with a descending pattern throughout the 28 days. TheraCal PT showed significantly lower Ca2+ release after 14 days compared to TheraCal LC.
Figure 2: Line chart and table representing calcium ion release results of Biodentine, TheraCal LC, and TheraCal PT.

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Surface characterization

Environmental scanning electron microscope with energy dispersive X-ray results

[Figure 3] reports the ESEM data and EDX analysis of Biodentine, TheraCal LC, and TheraCal PT immediately after setting. Biodentine displayed a uniform surface containing interspersed particles with a degree of porosity. EDX analysis showed high peaks of calcium, silica, and oxygen which are the main elements of tricalcium silicate. Chlorine from the aqueous calcium chloride, limited amounts of zirconia radiopacifier, and carbon from calcium carbonate were also detected. The set TheraCal LC and TheraCal PT specimens displayed mineral particles evenly distributed in the organic resin matrix resembling that of set resin composite. The EDX spectrum showed the presence of oxygen and carbon which are the characteristic elements of the organic phase. TheraCal LC showed calcium and silicon indicating the presence of calcium silicate particles. Silica from silicate glass was present in TheraCal PT together with limited amounts of fluoride and aluminum. No calcium was detected.
Figure 3: Environmental scanning electron microscope images with corresponding energy dispersive X-ray analysis for Biodentine, TheraCal LC, and TheraCal PT immediately after setting.

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ESEM data and EDX analysis of Biodentine, TheraCal LC, and TheraCal PT after 1-day storage are shown in [Figure 4]. Biodentine surface showed a degree of densification since porosities were filled with hydration products, calcium silicate hydrate, and calcium hydroxide. Few spherical precipitates were also detected on the surface. EDX analysis detected the presence of calcium, silicon, oxygen, and traces of phosphorous. TheraCal LC displayed few spherical precipitates on the surface. Calcium, silicon, carbon, and oxygen in addition to traces of phosphorous were detected. TheraCal PT showed no precipitates where carbon and oxygen were present as major components for resin matrix. Small traces of calcium, fluoride, and aluminum were detected.
Figure 4: Environmental scanning electron microscope images with corresponding energy dispersive X-ray analysis for Biodentine, TheraCal LC, and TheraCal PT after 1 day immersion in phosphate-buffered saline.

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Following 14 days storage of Biodentine, TheraCal LC, and TheraCal PT, globular deposits were detected covering the majority of Biodentine surface [Figure 5]. Calcium was detected and the level of phosphate increased considerably. TheraCal LC also showed an evident globular surface layer coating most of the specimen's surface as shown in [Figure 5]. EDX analysis revealed the presence of calcium, phosphate, and oxygen which might indicate apatite formation. TheraCal PT showed limited scattered spherical precipitates on the surface. Carbon and oxygen were present as major elements of the resin matrix with limited amounts of calcium and phosphorous.
Figure 5: Environmental scanning electron microscope images with corresponding energy dispersive X-ray analysis for Biodentine, TheraCal LC and TheraCal PT after 14 days immersion in phosphate-buffered saline.

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[Figure 6] shows ESEM data and EDX analysis of Biodentine, TheraCal LC, and TheraCal PT after 28 days. Both Biodentine and TheraCal LC showed a continuous layer of globular particles covering the entire surface of the specimen. Such layer is formed mainly of calcium, phosphate, and oxygen that might indicate apatite layer development. No silicon could be detected as the precipitated layer completely covered the discs' surfaces. TheraCal PT showed the presence of few precipitates on the surface. EDX analysis revealed the presence of oxygen and carbon as major constituents together with calcium and silicon.
Figure 6: Environmental scanning electron microscope images with corresponding energy dispersive X-ray analysis for Biodentine, TheraCal LC, and TheraCal PT after 28 days immersion in phosphate-buffered saline.

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Attenuated total reflectance/Fourier transform infrared analysis

[Figure 7] reports overlaid IR spectra for each of Biodentine [Figure 7]a, TheraCal PT [Figure 7]b and TheraCal LC [Figure 7]c both before immersion (control) and after 28 days of immersion in PBS.
Figure 7: The overlaid Fourier transform infrared spectra of control samples before immersion and after 28 days for Biodentine (a), TheraCal PT (b), and TheraCal LC (c).

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Fresh Biodentine samples presented bands attributed to O-Si-O (813 cm−1), and O-Si-O/Si-O-Si (524 cm−1, 455 cm−1). Silica group was also detected at 640 cm−1 in addition to a silanol group that might be part of the calcium silicate hydrate layer (CSH) at 956 cm−1.[21],[22],[23],[24] The carbonate group of calcite appeared at 875 cm−1 and about 1458 cm−1.[25] A band at 3,465 cm−1 indicated the presence of Ca(OH)2. A peak representing an aliphatic C = C (1638 cm−1) may be due to the presence of the hydrosoluble polymer present in the Biodentine liquid as a water-reducing agent.[22]

After storage, the peak intensity of Ca (OH)2 at 3,465 cm−1 decreased indicating the dissolution of Ca (OH)2 in PBS. Another peak for Ca (OH)2 also appeared at 1428 cm−1.[22]

The band assigned to calcium carbonate at 1458 cm−1 showed a reduction in peak intensity. PO43−groups formed bands at 560 cm−1 and 600 cm−1 in addition to a strong band at 1042 cm−1.[26],[27],[28]

Analyzing freshly set TheraCal LC and TheraCal PT, the presence of silicate was noticeable at 813 cm−1 and at 950 cm−1 for TheraCal LC, and 460 cm−1 for both types of cements. A very strong and broad band of silicate was also observed at 1107 cm−1 for TheraCal LC and for TheraCal PT at 1120 cm−1.[22] Stretching vibration of C = O and C = C appeared at 1720 cm−1/1725 cm−1 and 1638 cm−1/1640 cm−1, respectively, in TheraCal LC and PT spectra attributing to their polymeric content. Bands at 1511 cm−1/1513 cm−1 (C-H aromatic ring), 1250 cm−1 (C-O), 1300 cm−1 (C-H), and 1385 cm−1 (CH3) also indicate the presence of resin.

After 28 days of storage in PBS, PO43− groups formed bands at 560 cm−1, 600 cm−1 and 1100 cm−1.

The band at 3465 cm−1 assigned to the hydroxyl groups of Ca (OH)2 appeared in both types of TheraCal after storage indicating the commencement of the hydration reaction.[22]

Micro-Raman analysis

Raman spectroscopy

Raman spectra for Biodentine, TheraCal LC, and TheraCal PT both before immersion [Figure 8]a and after 28 days of immersion in PBS [Figure 8]b are shown in figure.[8]
Figure 8: The overlaid Raman spectra of Biodentine, TheraCal LC and TheraCal PT before immersion (a) and after 28 days (b).

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For freshly prepared specimens, [Figure 8]a indicates the presence of a band at 1085 cm−1 assigned to Ca silicate[29] which is of better intensity in Biodentine compared to TheraCal LC, while for TheraCal PT, this band appears rare and weak. The CaCO3 band at 1080 cm−1 was observed in Biodentine only.[30] Ca(OH)2 at 252 cm−1 and 356 cm−1 exhibited a medium band in Biodentine.[31] A C = C band appeared at 1609 cm−1 assigned to the organic content of both TheraCal types.[32]

After 28 days of immersion in PBS, the recorded spectra showed additional bands at 960 cm−1 and 1040 cm−1 assigned to (PO4)−3.[33] It is noteworthy that the (PO4)−3 band at 1040 cm−1 appeared in all cement types while that at 960 cm−1 appeared in Biodentine and TheraCal LC only. Ca (OH) 2 bands at 252 cm−1 and 356 cm−1 appeared in TheraCal LC and TheraCal PT after immersion in PBS.

Raman mapping

[Figure 9] shows the Raman mapping for Biodentine [Figure 9a], TheraCal LC [Figure 9b], and TheraCal PT [Figure 9c] at symmetric Raman band 1040 cm−1 attributed to phosphate (PO4)−3 both before immersion (control) and after 28 days of immersion. Regarding the nonimmersed samples, charge-coupled device (CCD) counts showed peaks of 2.8 maximum, indicating nearly no presence of phosphate. Most probably such color-coded peaks were due to noise during Raman analysis.
Figure 9: The Raman mapping of Biodentine (a), TheraCal LC (b), and TheraCal PT (c) for control samples and after 28 days immersion presenting the distribution of (PO4)−3 at 1040 cm−1.

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After immersion, Biodentine image showed major peaks in the cyan, green, and blue regions indicating about 9–15 CCD counts. The maximum concentration of the phosphate bond might reach 27 CCD counts represented by few yellow peaks. As for TheraCal LC, most of the peaks were in the cyan, blue, and green regions referring to ~5–6 CCD counts. A few distributed yellow peaks were present indicating ~15 CCD counts.

The maximum phosphate bond concentration for TheraCal PT stored samples was 9–10 CCD counts (yellow and orange peaks). Major peak intensities were within the blue and green region representing approximately 5–6 CCD counts. Phosphate concentration dispersed over the investigated areas thus seemed to be of lower CCD counts compared to Biodentine and TheraCal LC.

  Discussion Top

HCSCs have shown the capability of creating a satisfactory “bioactive” surface possessing a suitable architecture following immersion in various simulated body fluids. Such surface was established through calcium phosphates nucleation and the development of an apatite layer. HCSCs are a subject of numerous improvements and modifications.[1],[34] They exhibit variations in their setting modes including hydration reactions, light, and dual polymerization reactions. However, limited researches are present regarding some of these cements.[35] To the best of our knowledge, a single study was recently published regarding TheraCal PT bioactivity.[36] This study, thus, sought to investigate the bioactivity of dual-cured TheraCal PT as compared to light-cured TheraCal LC and Angelus MTA.

Ca2+ release together with alkalizing activity is the foundation for favorable biological properties of HCSC utilized in pulp therapy and regenerative endodontic procedures.[19],[37] The leached Ca2+ enhances the growth of human dental pulp cells in a dose-dependent manner and boosts the activity of pyrophosphatase, which aids in maintaining dentine mineralization and the development of a dentine bridge.[20],[38] Furthermore, the production of hydroxyl ions increases the pH of the surrounding environment causing pulpal tissue irritation. Superficial necrosis thus develops on exposed pulp, stimulating mineralization against the necrotic zone. Hydroxyl ions also encourage the production of alkaline phosphatase and bone morphogenic proteins, which take part in the mineralization process.[37] Accordingly, it was of prime importance to evaluate Ca2+ release and assess the pH of the leachates of different cements.

PBS was used in various researches mimicking the physiologic body fluids for evaluation of the in vitro bioactivity of Biodentine, TheraCal and MTA like materials. Consequently, PBS was selected as an immersion medium in this study.[18]

Biodentine exhibited the highest levels of Ca2+ release throughout all time intervals while both TheraCal PT and LC showed less Ca2+ release with no significant difference between them. These findings are consistent with those of previous studies stating that resin modified HCSCs showed limited bioactivity and reduced Ca2+ release compared to Biodentine.[16],[19],[20],[39],[40]

Compositional differences between Biodentine and TheraCal LC might be a plausible explanation for such Ca2+ release results. Biodentine powder is formed mainly of tricalcium silicate (80.1%) while its liquid contains a calcium chloride accelerator that functions as a water-reducing agent. Sources of calcium thus seem to be abundant in Biodentine composition.[16],[41]

Contrarily, TheraCal LC's safety datasheet stated that it comprises a resin matrix (10%–30%) formed mainly of polyethylene glycol dimethacrylate while portland cement Type III which acts as a calcium ion source constitutes a percentage that does not exceed 50% of the total composition. Such configuration indicates less Ca2+ sources in TheraCal LC compared to Biodentine. Similarly, the aforementioned explanation can be applied to TheraCal PT since it is a resin-modified calcium silicate cement consisting of polyethylene glycol dimethacrylate (10%–30%) and Bis-GMA (5%–10%) resin matrix as per its safety datasheet. It is noteworthy that the safety data sheet of TheraCal PT (base and catalyst) did not mention the presence of calcium by any means in its composition.

Another reason for higher Ca2+ release of Biodentine is its porous [Figure 3] extremely hydrophilic structure in which hydration occurs significantly producing large amounts of calcium silicate hydrate and calcium hydroxide.[16],[40]

On the contrary, the resinous structure of TheraCal LC and TheraCal PT lacks sufficient porosities to allow such hydration reaction to occur. They rely mainly on water sorption and diffusion within the hydrophobic resin matrix which is somehow limited. It could be postulated that deficient hydration reactions of TheraCal LC and TheraCal PT is due to the sparse moisture diffusion in the set resin material causing deficient Ca2+ release.[16],[42] Biodentine showed a significant drop in Ca2+ release at day 28; a similar pattern was detected in a previous study.[43]

Concerning alkalizing activity, Biodentine showed significantly higher alkalizing potential at all time intervals compared to both TheraCal PT and TheraCal LC and this goes in line with other similar studies.[21],[33] TheraCal LC alkalizing activity was significantly higher than that of TheraCal PT after 1 day; however, no difference was detected after 14 and 28 days.

Alkalizing potential of calcium silicate cements is due to the formation of calcium hydroxide that undergoes ionization when subjected to moisture producing hydroxyl ions.[3] Accordingly, an increase in Ca2+ release is suggestive of higher hydroxyl ion diffusion. Low solubility of TheraCal LC together with limited water sorption of its resin matrix hinders the hydration reaction of calcium silicate to calcium hydroxide; hence reducing its alkalizing activity.[14] Owing to its resinous nature, such postulations appear applicable as well to TheraCal PT's results.

pH results were further supported by ATR/FTIR analysis, where Ca (OH) 2 was detected in Biodentine both before and after immersion in PBS at 1428 cm−1 and 3,465 cm−1 and in both types of TheraCal at 3465 cm−1 only after storage. Raman analysis followed nearly the same trend where Ca (OH) 2 bands for both types of TheraCal appeared only after storage indicating less sources of hydroxyl ions in TheraCal.

All silicate cements showed a significantly increasing pattern in pH up till 14 days. Such increase in pH values with time has been observed previously.[44]

Calcium silicate-based cements are capable of developing hydroxyapatite precipitates when placed in contact with phosphate-containing physiological fluids.[45] This formed apatite coating enhances cell attachment, differentiation, and tissue repair associated with mineralized tissue synthesis.[46] Surface characterization of discs soaked in PBS solution at different time intervals was thus performed in the current study using ESEM/EDX analysis, Raman spectroscopy, and FTIR spectroscopy.

ESEM was used instead of the conventional SEM to avoid any surface preparation or coating of the discs before imaging. This prevented any change in their surface chemistry and allowed evaluation of the same disc at different time intervals as intended.[47] Raman and IR spectroscopy are corresponding techniques capable of detecting silicate, carbonate, hydroxyl and phosphate vibrational modes, representing the hydration process of the cement along with apatite formation.

ESEM of freshly set Biodentine showed a uniform surface containing interspersed particles. EDX displayed the presence of calcium (approximately 50%) from tricalcium silicate. Regarding TheraCal LC, EDX analysis at various points showed traces of calcium (1wt%), while in TheraCal PT no calcium was detected at any points of analysis [Figure 3]. Such findings are in line with calcium release results, where Biodentine showed the significantly highest values due to its higher calcium content. FTIR and Raman spectroscopy for freshly prepared specimens seemed to support EDX analysis where calcium hydroxide and calcium carbonate bands as Ca2+ sources were present in Biodentine but absent in both types of TheraCal [Figure 7] and [Figure 8]. Calcium silicate bands were common in Biodentine, TheraCal LC, and TheraCal PT, however for TheraCal PT, these bands were the fewest (460 cm−1, 1120 cm−1) in FTIR analysis and of very weak intensity in Raman analysis.

Storage of Biodentine and TheraCal LC discs for 14 and 28 days in PBS led to the formation of continuous layers of globular deposits on their surfaces. EDX analysis at various points of these layers revealed the presence of both calcium and phosphate with increased peak intensities [Figure 5] and [Figure 6] which might enhance the formation of an appetite layer.[48] However, TheraCal PT discs exhibited scarce precipitates on their surface with low calcium and phosphate percentages as detected by EDX. Such EDX analysis pattern was in line with that of Rodríguez-Lozano et al.[36]

Such findings were further confirmed via FTIR and Raman spectroscopy. FTIR spectra for stored Biodentine, TheraCal LC, and TheraCal PT showed bands corresponding to PO43−which are attributable to the presence of hydroxyapatite [Figure 7].[26],[27]

Moreover, peak heights of the phosphate bands in TheraCal PT at 560, 600, and 1100 cm−1 were approximately one third that of Biodentine and TheraCal LC [Figure 7]. As for Raman spectroscopy, bands at 960 and 1040 cm−1 assigned to (PO4)−3 were detected. TheraCal PT exhibited the weakest band intensity relative to Biodentine and TheraCal LC, thus failed to develop appropriate phosphate deposits on its surface [Figure 8]. It is worth noting that Theracal PT was not able to from (PO4)−3 bands at 960 cm−1 as Theracal LC and Biodentine did [Figure 8].

Raman mapping results at 1040 cm−1 were also in line with that of the other surface characterization methods where Biodentine samples exhibited the highest concentration of (PO4)−3 bonds followed by TheraCal LC while Theracal PT showed the weakest peak intensities for (PO4)−3 bonds with the least CCD counts distribution [Figure 9].

Although TheraCal LC showed reduced Ca2+ release compared to Biodentine, it was able to form surface deposits rich in calcium and phosphate as those of Biodentine. Limited moisture diffusion within the resin matrix of TheraCal LC[16] might have hindered its hydration reaction and Ca2+ release; however, it was sufficient to develop calcium and phosphate deposits on its surface.

Combined results of Ca2+ release and alkalizing potential pointed out the high bioactivity of Biodentine compared to TheraCal LC. However, TheraCal LC still developed a mineralized layer rich in calcium and phosphate despite its lower Ca2+ release. As for TheraCal PT, its limited Ca2+ release, low alkalizing potential as well as failure to develop adequate calcium phosphate deposits reflected its poor in vitro bioactivity. Based on the above findings, the null hypothesis was thus rejected.

One of the limitations of this study is the lack of clinical simulation, thus bioactivity results might appear higher than expected in clinical situation where availability of moisture is limited. However, evaluation of hydration and bioactivity of calcium silicate – based cements is usually performed in vitro in standardized conditions to allow data correlation.[40] Moreover, the novelty of TheraCal PT favors conducting the study initially outside the biological context to be able to compare it with other HCSCs. Another drawback is the absence of enough previous studies in the literature concerning TheraCal PT to confirm or reject our findings.

  Conclusion Top

Within the limitations of this study, it could be deduced that TheraCal PT possesses reduced in vitro bioactivity in terms of Ca2+ release, alkalizing potential and apatite formation compared to Biodentine and TheraCal LC. Biodentine exhibits potent in vitro bioactivity compared to TheraCal LC which shows potential bioactivity in the form of precipitate formation. Further laboratory investigations and clinical trials regarding bioactivity of these materials are still required.


The authors would like to thank Alex Dent Company, the official distributor of Bisco Inc. in Egypt for supplying the required materials from Bisco for free.

Financial support and sponsorship


Conflicts of interest

The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or nonfinancial in this article.

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]

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