Introduction
The pulp can be compromised by caries, trauma, or mechanical injury. Promoting reactive dentin formation and maintaining a healthy pulp are vital in immature permanent teeth with incomplete root development[1]. The application of materials to protect the pulp from exposed pulp or deep cavities is referred to as pulp capping; for successful pulp capping, the capping materials must protect the pulp from bacteria or mechanical forces while demonstrating biocompatibility[2-4].
Since Phillip Pfaff first performed pulp capping by placing small gold foils on the exposed pulp in 1756, various materials have been used for pulp protection. Over the past few decades, calcium hydroxide has been considered the “gold standard” and is widely used[5]. However, there have been reports of numerous tunnel defects in dentinal bridges after pulp capping using calcium hydroxide and its potential for causing microleakage due to dissolution over time[6].
Resin-modified calcium hydroxide materials like UltraBlend™ plus (UBP, Ultradent Products, Inc., South Jordan, UT, USA), which enable direct pulp capping, exhibit superior physical properties, such as low solubility and resistance to dissolution over time, in addition to being light-curable[7]. These materials have demonstrated lower cytotoxicity than the conventional two-paste self-curing calcium hydroxide cement[8].
Mineral trioxide aggregates (MTAs) have been developed to address the limitations of conventional calcium hydroxide materials. Compared to calcium hydroxide, MTA induces less necrosis and inflammatory responses in the pulp. MTA can form thicker dentinal bridges without tunnel defects and exhibits superior sealing ability and biocompatibility, surpassing the characteristics of calcium hydroxide[9].
However, owing to its long setting time and poor manipulability, MTA may have decreased marginal adaptability and potential microleakage[10]. In particular, an inhomogeneous mixing process may compromise the physicochemical properties of MTA, leading to increased solubility and porosity[11,12].
Various enhanced materials with modified ingredients have been developed to overcome these drawbacks. One such material is TheraCal LC™ (TLC, Bisco Inc., Schaumburg, IL, USA), a resin-modified light-curable calcium silicate material developed in 2011. The TLC column was packaged in a single syringe, allowing immediate use without a mixing process. Curing can be initiated through light curing or hydration and does not require additional dentin pretreatment[13,14].
Subsequently, TheraCal PT™ (TPT, Bisco Inc.), a dualcure resin-modified calcium silicate material capable of self-curing, was introduced to the market in 2019. TPT exhibited improved chemical properties compared to TLC[15]. According to the manufacturer, the TPT offers thixotropy, allowing precise placement in the deep cavity, as intended by the clinician. Restorative materials can be directly applied owing to their dual-cure functionality. However, studies on the physical and biochemical properties of TPT are limited.
Ease of manipulation and short setting times are crucial considerations[16,17]. Given these advantages, clinically widely used pulp-capping materials include UBP, a calcium hydroxide-based material, and TPT and TLC, both calcium silicate-based materials. All three materials were available in syringe form for immediate use and were light-curable. Research comparing the characteristics of pulp-capping materials that share these common features is currently insufficient. This study aimed to compare the physical and biochemical properties of the newly introduced material TPT in comparison to its predecessor, TLC, and the calcium-hydroxide-based material UBP.
Materials and Methods
1. Physical properties
1) Sample preparation
The following light-curable pulp capping materials were selected for this study (Table 1): Ultra-blend™ Plus (UBP; Ultradent Products Inc.), TheraCal LC™ (TLC; Bisco Inc.), and TheraCal PT™ (TPT; Bisco Inc.). Each material was polymerized according to the manufacturer’s instructions with an LED light curing unit (B&LiteS, B&L Biotech, Ansan, Korea). UBP was first polymerized into a layer of 0.5 mm thickness, followed by 2 mm increments, curing between each increment. TLC was polymerized for 20 seconds in each layer to a thickness not exceeding 1 mm. And TPT was polymerized for 10 seconds.
2) Solubility
The solubility experiments were conducted following a modification of the ISO 6876 standard. Each specimen was prepared using a Teflon mold with an internal diameter of 20 mm and a height of 1.5 mm. After polymerization, the weight of each material was measured three times with a precision of 0.001 g, and the average value was recorded. Two specimens were placed in a shallow dish without contact or movement. Subsequently, 50 mL of distilled water was added, and the dish was covered. The dishes were placed in a cabinet at 37°C with a humidity of over 95% for 24 hours. The specimens were then removed. The specimens were rinsed with 2 - 3 mL of distilled water, and the rinsed water was collected in the original shallow dish. The dish was dried without boiling, allowing the water to evaporate, and then it was dried to a constant weight at 110°C. The dish was then cooled to room temperature. Subsequently, the difference between the initial and final weights of the shallow dish measured with a precision of 0.001 g was recorded as the amount of material leached from the specimen. The difference in weight was recorded as a percentage of the sum of the original weights of the two specimens, with an accuracy of 0.1%. This process was repeated three times for each material at a one-week interval, and the average was recorded as the solubility.
3) Water absorption
Water absorption experiments were conducted as previously described[18]. Each specimen was prepared using a Teflon mold with an internal diameter of 20 mm and a height of 1.5 mm, resulting in 10 specimens for each. The specimens were immersed in 25 mL of distilled water for 24 hours and stored in a cabinet at 37°C with a humidity over 95%. Subsequently, the moisture on the specimen surface was removed using damp gauze. The weight of the moistened specimen was measured with a precision of 0.001 g. The specimens were dried until a constant weight was achieved at 37°C and remeasured. The difference in weight was recorded as a percentage of the final weight with an accuracy of 0.1%. Each weight was measured three times, and the average value was used.
4) Dimensional change
A dimensional change experiment was conducted according to a previously described protocol[19]. Each specimen was prepared using a Teflon mold with an internal diameter of 4 mm and a height of 6.04 mm, resulting in 10 specimens for each. After immersing the specimens in 6.72 mL of distilled water for 30 days, they were stored in a cabinet at a temperature of 37°C. The specimens were then dried to a constant length. The internal diameter and height of each specimen were measured before and after storage and drying. The difference in length and volume was recorded as a percentage of the initial length and volume with an accuracy of 0.1%. Each length and volume were measured three times, and the average value was used.
2. Biochemical properties
1) Eluate preparation
Elution was conducted according to the standard surface area and extraction fluid volume table of ISO 10993- 12. Each specimen was prepared using a Teflon mold with an internal diameter of 16 mm and a height of 1 mm, resulting in 10 specimens for each. The surface area was 9.048 cm², and each specimen was immersed in 3.016 mL of distilled water at a ratio of 3 cm²/mL. The 10 specimens were stored in a shaking incubator (120 rpm) at 37°C each. The elution fluid was continuously extracted from the same set of specimens at 3 hours, 24 hours, 7 days, and 14 days of storage using a micropipette and at each time point, all the water was extracted and replaced with 3.016 mL of fresh distilled water. 28 days eluate was also extracted. The extracted elution fluid was filtered through a 0.2 µm syringe filter (Corning Inc., Corning, NY, USA).
2) pH test
The pH test was conducted using the elution fluid extracted at 3 and 24 hours, 7, 14, and 28 days. pH was measured using a calibrated pH meter (Orion VERSA Star Pro, Thermo Fisher Scientific, Waltham, MA, USA) at pH 10.0, 7.0, and 4.1. Before each measurement, the electrode was washed with distilled water and dried using gauze. Each elution fluid was measured three times, and the average value was recorded.
3) Ion release analysis
Ion-release analysis was performed using inductively coupled plasma atomic emission spectrometry (ICP-AES). Ion release analysis was conducted using the elution fluid extracted at 3 and 24 hours, and 7, 14, and 28 days. Approximately 3 mL of the elution fluid, filtered through a syringe filter, was mixed with 30 µL of concentrated nitric acid (HNO3; Wako Pure Chemical Industries, Osaka, Japan). The ion release quantities of calcium (Ca), sulfur (S), strontium (Sr), and silicon (Si) were measured using ICP-AES (OPTIMA 8300, Perkin-Elmer, Waltham, MA, USA). Each elution fluid was measured three times, and the average value was recorded.
4) Cell proliferation and viability assay
Human dental pulp cells (hDPCs; passages 4 - 5) were used in this study. The teeth from which the hDPCs were extracted were used for clinical purposes at Dankook University Dental Hospital following the guidelines approved by the Ethical Committee of the Institutional Review Board of Dankook University Dental Hospital (IRB number DKDUH 2023-06-003). One participant provided informed consent to participate in the procedure. hDPCs were seeded on a 96-well plate (SPL Life Sciences, Pocheon, South Korea) at a density of 1000 cells/well with 200 μL of DPSC growth media consisting of αMEM (Wellgene, Kyungsan, Korea) as the base medium, 20% Fetal Bovine Serum (Corning Inc., Corning, NY, USA), 1% Penicillin-Streptomycin (Gibco, Waltham, MA, USA), 1% Glutamax (Gibco), 0.1% 2-mercaptoethanol (Gibco). Cells were incubated for 24 hours under standard conditions (37°C, 5% CO2) for stabilization. At the 24-hour mark, the medium was discarded, and the eluate collected over 24 hours and 14 days, diluted with growth media (1 : 10, 1 : 2), was added to each well (five wells per material extract group). The dilution ratios commonly employed in previous studies that conducted similar cell experiments were selected[20-22]. Additionally, to diversify the concentration of materials reaching the cells, we utilized two different dilution ratios. For the control group, fresh hDPCs growth media were replenished in lieu of extract solution. After an additional 24 hours of incubation with the extract, it was replaced with a 10% CCK-8 solution (dissolved in growth media; 150 μL per well; Dojindo, Kumamoto, Japan) for 2 hours of incubation, concealed from light exposure. A portion of the CCK solution from each well was removed and transferred to a new 96-well plate, which was then inserted into a microplate reader (Varioskan LUX, Thermo Fisher Scientific) for absorbance assay (450 nm). Baseline optical density values were obtained from blank wells (CCK solution without cells) and were used for subsequent analyses.
Cell survival ratio was examined by Live and Dead staining (0.5 µM calcein AM and 2 µM ethidium homodimer-1 solutions, Thermo Fisher Scientific) on the DPSCs from above. Images were obtained using an optical microscope (IX71, Olympus, Tokyo, Japan).
All the experiments described were conducted in independent triplicates.
3. Statistical analysis
Statistical analyses were performed using the Kruskal- Wallis and post-hoc Mann-Whitney U tests with Bonferroni correction with IBM SPSS 21.0 (SPSS Inc., Chicago, IL, USA). The threshold of p-value in Bonferroni correction was 0.00833 (0.05 / 6) for the cell proliferation test as compared to four groups, including the control group. In other analysis, where we compared three groups (UBP, TLC, and TPT), the threshold p-value was 0.01667 (0.05 / 3).
Results
1. Physical properties
1) Solubility
The results of the solubility experiments are shown in Fig. 1 and Table 2. UBP, TLC, and TPT all satisfied the solubility criteria and were lower than the 3% required by ISO 6876. UBP exhibited the highest solubility with 2.68 ± 0.26%, followed by TLC and TPT with 0.23 ± 0.04% and 0.09 ± 0.02%, respectively. The solubility values of UBP and TPT differed significantly (p < 0.05).
2) Water absorption
The results of the water absorption experiments are presented in Fig. 2 and Table 2. TPT demonstrated the lowest water absorption rate at 1.13 ± 0.10%, followed by UBP and TLC with 6.28 ± 0.31% and 7.38 ± 0.62%, respectively. Only the water absorption rates of the TPT and TLC groups were significantly different (p < 0.05).
3) Dimensional change
The results of the dimensional change experiments are presented in Fig. 3 and Table 2. TLC exhibited the highest values in both linear and volumetric changes, with 7.79 ± 1.02% and 23.46 ± 1.89%, respectively (p < 0.05). There was no significant difference in the linear change rates between the UBP and TPT.
2. Biochemical property
1) pH
The results of the pH experiments are presented in Fig. 4 and Table 3. The pH of UBP’s elution fluid at 3 hours was 8.09 ± 0.33, indicating slight alkalinity; however, by the 28th day, it showed a value close to neutrality at 7.41 ± 0.07. Throughout all periods, the pH of UBP was lower than that of TPT and TLC (p < 0.05). At 24 hours, the pH of the TLC was higher than that of the TPT (p < 0.05); however, no significant difference was observed from 3 hours to 7 days onward (p > 0.05).
2) Ion release analysis
The results of the ion release analysis are shown in Fig. 5 and Table 4. UBP exhibited a cumulative calcium ion release of over 3000 ppm on the 28th day; however, it showed minimal release of Si, S, and Sr ions. TLC showed higher release of S, Sr, and Ca ions than TPT for all periods (p < 0.05); however, there was no significant difference in silicon ion release between TPT and TLC on the 28th day (p > 0.05).
3) Cell proliferation and viability assay
When the absorbance values of the control group were normalized to 100%, the cell proliferation rates for each day of elution with the elution fluids used are shown in Fig. 6 and Table 5,. At a dilution ratio of 1 : 2, the proliferation rate of TLC cells was significantly lower than that of UBP cells (p < 0.05). At a dilution rate of 1 : 10, when using the 24-hour elution fluid, the cell proliferation rate of TLC was higher than that of the control group (p < 0.05). When evaluating the cell proliferation rates for each day of elution fluid for all materials, using the 14- day elution fluid resulted in lower cell proliferation rates than using the 24-hour elution fluid (p < 0.05). The cell viability results after Live and Dead staining are shown in Fig. 7. Dead cells were observed in the staining results of the elution fluid diluted 1 : 2 for TLC.
Discussion
Pulp-capping materials play a crucial role beyond merely sealing the pulp; they contribute significantly to the regeneration of the dentin-pulp complex. These materials must possess appropriate biocompatibility, biological activity, and mechanical and physical properties to fulfill this role effectively[23-25]. Because there is no perfect material in all aspects, it is essential to compare and analyze the characteristics of the materials. This process helps to choose a more suitable pulp-capping material based on specific requirements and considerations. For example, an appropriate water absorption rate is essential for ion release[26]. However, high water absorption, which leads to high solubility, can induce porosity in the restoration or compromise the strength[27]. The dimensional change in the material should fall within an appropriate range, and the material should exhibit biocompatibility. Additionally, in pediatric dentistry, where moisture control can be challenging[28], materials with short setting times and easy manipulability are required.
Low solubility plays a crucial role for a pulp capping material to fulfill its role in providing a permanent seal against bacteria. One of the drawbacks of conventional calcium hydroxide-based materials is their high solubility and subsequent dissolution in tissue fluids[29]. In this study, UBP met the 3% criterion specified in ISO 6876; however, it exhibited a relatively high solubility. In contrast, the solubility of TPT was significantly lower than that of UBP (p < 0.05). The water absorption rate can be influenced by solubility; generally, a low water absorption rate indicates low solubility[30]. In this study, TPT and UBP exhibited water absorption rates that were proportional to solubility, whereas TLC’s water absorption rate did not correlate with solubility. The hydrophilic components of UBP promote water absorption[31], while the hydrophilic resin of TLC assists water absorption and initiates the hydration reaction of Portland cement[18]. The lower solubility value exhibited by TLC compared to that of UBP, yet similar water absorption rates, are thought to be attributed to the characteristics of TLC. This aligns with the findings of previous studies[18,32].
To recover pulp, pulp capping materials must possess dimensional stability[33,34]. In this study, the linear dimensional change rates of UBP and TPT did not show statistically significant differences (p > 0.05), whereas the linear dimensional change rate of TLC was the highest among the three materials (p < 0.05). This may be due to the hydrophilic resin content in TLC[13,35]. Although there are no specific standards for the dimensional change in pulp capping materials, if a material contracts significantly, it may create gaps that allow the passage of bacteria. Conversely, excessive expansion can lead to fracture of the surrounding tissues or restorative materials[36,37]. The volumetric expansion of resin-modified materials and their subsequent penetration into the dentinal tubules may increase the adhesive strength between the material and dentin[35]. However, because hygroscopic expansion does not always fill the gaps between the material and dentin[38], further research is needed to fully understand the implications of these characteristics in pulp capping materials.
The ability of pulp-capping materials to release hydroxyl and calcium ions is a crucial factor in their biological activities. In particular, hydroxyl ions induce tissue regeneration, exhibit antimicrobial effects, and stimulate the release of various proteins and growth factors, thereby promoting the recovery of dental tissues[39,40]. In this study, although UBP is a calcium hydroxide-based material, it exhibited a pH close to neutral. This suggests the presence of components that inhibit the release of hydroxyl ions[41]. Calcium ions assist in the differentiation and proliferation of osteoblasts, stimulate the expression of fibronectin genes in the pulp tissue, and induce the formation of hard tissues[42,43]. In a previous study using mouse primary osteoblasts, calcium ion concentrations ranging from 2 - 6 mM (approximately 80 - 240 ppm) were suitable for cell survival and differentiation. Slightly higher concentrations (6 - 10 mM) induced both differentiation and mineralization. However, concentrations exceeding 10 mM exhibited cytotoxic effects[44]. Furthermore, a study using hDPCs suggested that high concentrations of calcium ions could affect the genes related to bone formation. However, such high concentrations may inhibit alkaline phosphatase activity, suppress the COL1 gene, and be unbeneficial for cell proliferation and matrix formation. Therefore, optimizing calcium ion concentration in dental restorative materials is necessary[45]. In this study, TPT and TLC showed significantly lower calcium ion release than UBP did. However, the sustained calcium ion release from TPT and TLC for up to 28 days remained within the range that could promote the activity of boneforming cells and dental pulp cells[44,46]. In addition to hydroxyl and calcium ions, various other ions can influence the surrounding tissues. Sulfur ions exhibit antimicrobial properties[47,48] and strontium ions, which aid in calcification during bone formation, can induce the differentiation of mesenchymal stem cells into osteoblasts. Strontium ions induce odontoblastic differentiation in hDPCs and negatively affect the osteoclast activity of osteoclasts[49-51]. Silicon ions stimulate osteoblasts, aiding in the formation of new bone. As an element in dental restorative materials, silicon is believed to promote mineralization[52,53]. In this study, UBP exhibited high concentrations of calcium ions, whereas other ions were scarcely released. TLC showed a higher release of calcium, strontium, and sulfur ions than TPT at all the time points. However, there was no significant difference in the silicon ion concentration in the 28-day-released solution. The hydroxyl ion release from UBP was lower than that from TPT and TLC at all time points (p < 0.05), and from 3 hours to 7 days, there was no significant difference in hydroxyl ion release between TPT and TLC (p > 0.05). This result is consistent with previous studies[54,55]. The ion release capacity is influenced by water absorption and solubility[26]. In this study, the generally lower ion release observed with TPT than with TLC was likely a result of these factors.
TLC has been researched both in vitro and in vivo since its introduction to the market. However, many studies have recommended limiting its use to indirect pulp treatments[15]. While there haven’t been many studies conducted yet, some research suggests that TPT exhibits better biocompatibility and bioactivity than its predecessor, TLC, to the extent that it can be compared to MTA[56]. This highlights the importance of evaluating the biocompatibility of hDPCs as pulp-capping materials, which, if lacking biocompatibility, may lead to a loss of vitality in the dental pulp[57,58]. In this study, we employed an indirect contact method between cells and materials using eluates of the materials. This method was selected because it can potentially induce cell growth more effectively than direct contact methods[59]. According to the literature, the standard practice is to use eluates for 24 hours. However, to assess the long-term effects of dental restorative materials, we added eluates for 14 days. Additionally, dilution ratios of 1 : 2 and 1 : 10 were used to account for potential variations in the material concentrations reaching the dental tissues[60]. In this experiment, when using a high dilution ratio (1 : 10) with a 24-hour eluate exposure, all materials, except for the eluate from TLC, exhibited cell viability similar to that of the control group. However, at a low dilution ratio (1 : 2), UBP and TPT showed higher cell viability than the control, whereas TLC showed lower cell viability than the control. This was confirmed by the Live and Dead staining images shown in Fig. 7. The results of this study are consistent with those of previous research, indicating that TPT does not exhibit cytotoxicity and supports cell proliferation[61]. Additionally, the results aligned with previous cell toxicity experiments based on the dilution ratios of TPT and TLC[15]. According to previous studies that described the potential for low cell survival rates with TLC, this effect could be attributed to the unpolymerized monomers present in the TLC[7,62,63]. This suggests that the self-polymerization characteristics of TPT may be advantageous compared with those of TLC. When comparing cell viability throughout eluate exposure, a difference in cell survival between 24-hour eluate and 14-day eluates was observed at a dilution ratio of 1:10; however, no significant difference was observed at a dilution ratio of 1 : 2.
This study has several limitations. First, the number of materials tested and specimens was insufficient. Additionally, as all experiments were conducted in vitro, the reaction with actual pulp tissues could not be determined. In particular, with the use of only dilution ratios of 1 : 2 and 1 : 10, as well as the analysis of only a 24-hour and 14-day culture in the cell proliferation experiments, further studies should investigate whether values such as pH, ion release, solubility interact with each other. Finally, other physical, biochemical properties and in vivo studies should be conducted.
Conclusion
The three restorative materials with easy handling and immediate applicability, owing to their syringe-type and light-curing capability, were compared in this study. The calcium silicate-based materials, TPT and TLC, showed lower calcium ion release than the long-used calcium hydroxide-based material, UBP. However, these values were within the range required to promote cell differentiation. Simultaneously, they showed higher S, Si, Sr, and OH- ion release than UBP. TPT exhibited lower water absorption, solubility, and volumetric dimensional changes than UBP and TLC. In cell experiments, TLC showed cytotoxicity at a 1 : 2 dilution ratio, whereas UBP and TPT demonstrated excellent cell survival rates across all dilution ratios and extraction periods. In summary, when considering using light-cured resin-modified pulp-capping materials in pediatric dentistry, calcium silicate-based materials may be an alternative to calcium hydroxide-based materials. Based on a comprehensive analysis of the physical and biochemical properties, TPT could be the preferred choice over TLC.
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