Calcium Release from Different Toothpastes after the Incorporation of Tricalcium Phosphate and Amorphous Calcium Phosphate

This study aimed to investigate the free calcium released from different brands of toothpaste after incorporation with a beta-tricalcium phosphate (β-TCP)/amorphous calcium phosphate (ACP) mixed powder and with β-TCP powder alone. Four brands of toothpaste were used for the experiment: Nano-Bio Activation Toothpaste, Colgate Total Advanced Whitening Toothpaste, BORONIA Herbal Whitening, and BioMin F. The investigated β-TCP and ACP powders were prepared by a two-step sintering method using raw coral material. Analytical results found that the mean calcium concentration of the β-TCP/ACP (2:1) powder in deionized water was 3.4% when the pH was between 5 and 9. Moreover, statistical results revealed that the toothpaste containing β-TCP powder had significantly higher calcium concentrations than the normal toothpaste. The toothpaste containing mixed β-TCP/ACP powder had a higher calcium concentration than the toothpaste containing only β-TCP powder. Twice as much free calcium could be released from β-TCP/ACP toothpaste than from β-TCP-only toothpaste. Thus, toothpaste containing β-TCP/ACP mixed powder offers greater benefits to facilitate the remineralization of enamel than toothpaste containing only β-TCP.


Introduction
The human oral environment undergoes a dynamic process of demineralization and remineralization every day. The oral environment changes from neutral to acidic due to metabolites produced by bacteria in the mouth. An acidic oral environment causes demineralization of the tooth surface, leading to caries, periodontal disease, and tooth sensitivity. In the demineralization process, calcium phosphate (CaP) crystals on the surface of teeth are dissolved by the acidic environment, causing a loss of calcium ions. However, the oral environment can be made to return to neutral by performing oral cleaning, thus halting tooth surface demineralization. Subsequently, the free calcium and phosphate in the saliva will re-enter the tooth surface to form a protective layer of CaP crystals in a remineralization process [1][2][3]. Therefore, calcium ions are a crucial element of the oral environment since their presence can promote the formation of CaP crystals inside the teeth during the remineralization process.
It is well known that the outer enamel of human teeth consists of micron-sized calcium carbonate (CaCO 3 ) deficient hydroxyapatite (HAP) microcrystals [4]. The main component of the outer enamel is HAP (around 97%) [5,6]. Recent studies have found that CaP, such as amorphous calcium phosphate (ACP), beta-tricalcium phosphate (β-TCP), and alpha-tricalcium phosphate (α-TCP), are similar to the natural enamel [7][8][9]. Among all compounds of CaP crystals, β-TCP and ACP are the most commonly used in oral care applications [10,11]. Although most CaP additives need to release calcium ions under acidic conditions, β-TCP can provide the best calcium ion release effect even in a neutral pH environment [12]. Accordingly, β-TCP is regarded as a stable tricalcium phosphate at room temperature. Hemagaran et.al. [12] also demonstrated that using β-TCP can increase calcium levels in saliva and promote the remineralization of tooth surface. In addition, it was also found that ACP presented as a unique artificial HAP [4]. Previously published studies have shown that ACP has good bone conduction and biodegradability, enhances cell proliferation ability, and promotes cell adhesion [13,14]. Moreover, ACP and its composites are considered to be an effective remineralization agent that is often used for remineralization in the dental fields [15][16][17][18].
As stated above, an adequate increase in the amount of free calcium and phosphate in the oral environment plays an important role in facilitating tooth surface remineralization during oral cleaning. From an oral care point of view, it would be beneficial if daily use toothpastes could be developed with the addition of various remineralization agents to prevent the formation of caries without sacrificing any useful properties. Hence, the present study reports on the synthesis and characterization of β-TCP and ACP derived from coral-based raw material through a two-stage sintering method. We aimed to investigate the concentration of calcium in toothpaste incorporated with β-TCP and ACP powders. We hypothesized that adding β-TCP and ACP to toothpaste would increase the release of free calcium in the oral environment, which might be potentially beneficial for the tooth remineralization process.

Sample Sintering
The raw coral materials were provided by Popeye Marine Biotechnology Limited (New Taipei City, Taiwan). A two-step sintering method was performed using a heating furnace. For the TCP powder, specimens were first heated for 1 h with a 3 • C/min ramp rate to 800 • C, then heated again to higher temperatures (1100 and 1200 • C). Afterwards, specimens were soaked with a 5 • C/min ramp rate for 1, 2, and 3 h, respectively. For the ACP powder, the specimens were obtained through the precipitation method. In the first sintering process, the specimens were maintained at 700 • C; afterward, the specimens were continually heated at higher temperatures above 800 and 900 • C. Finally, the specimens were soaked with a 5 • C/min ramp rate for 1 h.

Characteristics of the Sintered TCP and ACP Powders
Powder X-ray diffractometry (XRD; Model 2200, Rigaku Co., Tokyo, Japan) was used to identify the crystallinity of the untreated TCP and ACP. For excitation, monochromatic Cu Kα radiation was generated, operating at 40 kV and 30 mA. The XRD data were taken over a 2θ range of 20 • -45 • at the account time of 5 s and a step size of 0.04 • /step. All of the corresponding peaks were analyzed according to the database from the Joint Committee on Powder Diffraction Standards (JCPDS). The different numbers of peaks represent the characteristic peaks of different samples. The Fourier-transform infrared (FTIR) spectra of sintered TCP and ACP powders were obtained in diffuse reflection mode through the potassium bromide pellet method. The FTIR spectra obtained were in the range of from 400 to 4000 cm −1 (Perkin Elmer Inc., Waltham, MA, USA). An NR-1800 Raman spectrophotometer (JASCO, Tokyo, Japan) was used to measure the Raman spectra of sintered TCP and ACP powders using charge-coupled device detector and an exposure time of 4 s.

Preparation of Samples for pH Measurements
The pH value of the investigated samples was measured by a pH meter (PB-10 pH meter, Sartorius AG, Goettingen, Germany). As much as 1 g of the sample was weighed out into a 100 mL beaker, deionized water was added to the mark, and then the sample was dispersed for 1 h by using a magnetic plate. The pH meter was then calibrated and inserted into the beaker. Finally, the pH meter readings were recorded.

Preparation of TCP/ACP Powders Mixed Toothpaste
The TCP powders calcined at 1100 • C for 1 h were mixed in a 2:1 ratio with the ACP powder calcined at 900 • C for 1 h. Afterward, the mixed powder was added to each gram of toothpaste in ratios of 10% and 25%, and then, the mixture was stirred evenly for testing. Four brands of toothpaste were used in this study: Nano-Bio Activation Toothpaste (Pac-Dent Inc., Brea, CA, USA), Colgate Total Advanced Whitening Toothpaste (Colgate-Palmolive Company, New York, NY, USA), Boronia Herbal Whitening (BORONIA, Taichung, Taiwan), and BioMin F toothpaste (BioMin Technologies Limited, London, UK). The ingredients of the investigated toothpastes are listed in Table 1. The effect of free calcium released after the TCP and TCP/ACP powders were incorporated with the toothpastes was then evaluated.

Measurement of Calcium Release
The calcium concentration of each sample was measured by ethylenediaminetetraacetic acid (EDTA, Sigma Aldrich, Taipei, Taiwan) titration. At first, as much as 1 g of the mixed powder or 2 g of the powder mixed with toothpaste was weighed out accurately using an Erlenmeyer flask; then, deionized water was added, and the mixture was stirred for about 1 h on a magnetic plate until it was well-dispersed. Afterwards, the sample was centrifuged for about 30 min until a clear supernatant was obtained. Following this procedure, 10 mL of the supernatant was pipetted into a 250 mL of Erlenmeyer flask. Then, 20 mL of deionized water and 20 mL of NH 3 ·H 2 O-NH 4 Cl buffer solution were added to bring the pH to 10.0. After the solution was mixed well, 2 drops of Eriochrome Black T indicator (Sigma Aldrich, Taipei, Taiwan) were added, and the flask was swirled. Finally, 0.01 M EDTA was titrated to the blue endpoint, at which point the volume recorded was equal to the difference between the final endpoint and the starting point, measured in mL.

Statistical Analysis
SPSS 19.0 software (SPSS Inc., Chicago, IL, USA) was used to conduct the statistical analysis. In order to compare the calcium concentration between different brands of toothpaste, an ANOVA test was conducted. The value obtained was considered statistically significant with a p-value < 0.05.  Figure 1a shows the XRD patterns of the untreated TCP. We observed that the raw material resembled the structure of CaP closely, indicating that TCP accounted for most of the TCP/HAP biphasic coral powder. According to the (0210) plane in the XRD pattern, the TCP sample can be identified as β-TCP. In addition, based on JCPDS card 00-009-0169, the result was a typical tricalcium phosphate structure, and its quality was indexed. Furthermore, no additional diffraction peaks were found, indicating that the untreated TCP had a single tricalcium phosphate structure. Figure 1b shows the XRD patterns of the untreated ACP. The results show a broadband of ACP presence in the XRD pattern. Moreover, a weak peak of Ca 10 (PO 4 ) 6 (OH) 2 was also found, with the characteristic peak of the (211) plane as a reference. is not visible, indicating that most of the raw materials were β-TCP. (b) A broad diffraction peak, which is characteristic of the amorphous state of ACP (blue line). Only one peak corresponding to 211 of the HAP (black cube) exists, indicating that most of the raw materials were ACP. The x-axis of the spectra is the defined diffraction angle between the incident and the reflected x-ray; the y-axis is the extent of the crystallinity of the particular plane. Figure 2 highlights the effect of different heat treatment temperatures and heat treatment times on the sample structure. FTIR spectra were used to test the TCP calcined at 1100 • C for 1, 2, and 3 h, the TCP calcined at 1200 • C for 2 h, and the ACP calcined at 800 and 900 • C for 1 h. It was found that the two bands at 1030 and 560 cm −1 could be assigned to the stretching (P-O) and the deformation (O-P-O) vibration modes of PO 4 3− , respectively. The raw materials of both the TCP and the ACP showed broad peaks with a less resolved feature for the ACP. The characteristic peak of PO 4 3− at 1100 cm −1 was enhanced significantly after sintering, which indicates that the structure of the ACP was changed to α-TCP and β-TCP at 800 and 900 • C. After the heat treatment, some fine peaks were resolved in the TCP and ACP spectra, suggesting an increase in molecular movement. The appearance of the TCP 1200 • C/2 h became similar to that of the intact TCP. Moreover, the fine peak positions (630, 600, and 570 cm −1 ) for the deformation vibration mode were found to be almost the same for the both the heated TCP and the ACP, while those for the stretching vibration mode looked different. A very sharp peak at 1138 cm −1 was found only in the 1100 • C-heated TCP spectra. FTIR spectra of (a) sintered TCP and (b) sintered ACP powder. The results from (a) show that TCP can be generated stably at a temperature of 1300 • C. The results from (b) indicate that the structure of ACP was converted into TCP after heat treatment. The x-axis of the spectra is the frequency of infrared light, or wave numbers, measured in reciprocal centimeters (cm −1 ); the y-axis is the infrared light transmittance. Figure 3a,b display the Raman spectra of the sintered TCP and ACP samples. The characteristic peaks at 960 cm −1 , which were assigned to the symmetric stretching mode of PO 4 3− , appeared in both the TCP and the ACP samples after sintering. In addition, when the treatment temperature (TCP 1 h, ACP 2 h) or the heat treatment time was increased (for TCP 1100 • C), the peak shifted to a higher wavenumber (ν) and the bandwidth at its half-height (W 1/2 ) became smaller. This finding indicated that compressive stress was imparted to the PO 4 3− moiety due to some internal compaction, and crystallinity increased (Figure 3c,d). Figure 3. Raman spectra (a,b) and crystallinity analysis (c,d) of the sintered TCP and ACP shows that heat treatment not only promoted the formation of TCP and converted ACP into TCP but also increased crystal strength. The x-axis of the Raman spectra is the frequency of light measured in reciprocal centimeters; the y-axis is scattered light. Figure 4 illustrates the free calcium concentration of 1 g of TCP/ACP mixed powder at different pH levels. It was observed that the TCP/ACP mixed powder showed a stable amount of free calcium release with an average concentration of 3.4% in the pH 5 to pH 9 range. This range meets the pH requirements for manufacturing a toothpaste. In addition, the calcium concentration increased significantly to 5.0 and 6.2 at pH 10 and pH 11. Although a higher free calcium release was noticed at pH 10 and 11, we assume that it may have been caused by the calcium oxide remaining from the incomplete sintering process of some of the raw material. Figure 5a depicts the free calcium concentration of different brands of toothpaste containing TCP powder. It was found that the mean calcium concentrations for 1 g of each toothpaste without TCP powder was 0, 0, 0.10, and 0.11, respectively. The mean calcium concentration obtained from 1 g of each toothpaste containing the 10 wt% TCP powder was 1.38 ± 0.10, 1.56 ± 0.21, 1.26 ± 0, and 1.69 ± 0.21, respectively. In addition, the mean calcium concentration of 1 g of toothpaste containing the 25 wt% TCP powder was 2.14 ± 0.07, 2.39, 1.69 ± 0.17, and 1.69 ± 0.17, respectively. The toothpaste containing TCP powder had a significantly higher calcium concentration than normal toothpaste. Figure 5b shows the free calcium concentration of different brands of toothpaste containing the TCP/ACP mixed powder. The mean calcium concentration for 1 g of each normal toothpaste was 0, 0, 0.1, and 0.11, respectively. The mean calcium concentration obtained from 1 g of each toothpaste containing the 10 wt% TCP/ACP mixed powder was 1.8, 1.8, 1.8, and 1.8, respectively. Moreover, each 1 g of toothpaste containing the 25 wt% TCP/ACP mixed powder had a mean calcium concentration of 2.79 ± 0.17, 2.79 ± 0.17, 3.09 ± 0.17, and 2.69 ± 0.30, respectively. The toothpaste containing the TCP/ACP mixed powder had a higher calcium concentration than the toothpaste containing only the TCP powder.\   The toothpaste containing only TCP powder had a significantly higher calcium concentration than the normal toothpaste. Furthermore, the toothpaste containing the TCP/ACP mixed powder had a higher calcium concentration than the toothpaste containing only TCP powder.

Discussion
In the present study, we prepared TCP and ACP powders from coral-based raw material and added the powders into commercially available toothpaste in concentrations of 10% and 25%. The quantity of free calcium released from the toothpaste was then measured. It was found that the raw material had almost completely changed from a CaCO 3 structure to a CaP structure by means of a two-step sintering method. It is known that the raw material made from coral is mainly comprised of CaCO 3 and calcium oxide (CaO) and that CaCO 3 and CaO are the main chemical compounds supporting the bone and tooth remineralization process [7,19]. Previously published studies indicated that high temperature and high pressure could replace the CaCO 3 of coral with HAP or TCP, which is a process known as replamineform [20,21]. In accordance with the previous studies, here, we found that high temperature and high pressure promoted the conversion of HAP to TCP following the previous chemical reaction. We also found that the structure of the ACP was changed to α-TCP and β-TCP at 800 and 900 • C [22,23]. A similar result was also seen in previous studies discovering that ACP could be converted to α-TCP and β-TCP after sintering at 800 and 900 • C [24,25].
We also found that the mixed TCP and ACP powders released a stable amount of free calcium in the pH 5 to pH 9 range. A previous study demonstrated that pH has a significant effect on ACP precipitation during the synthesis process, in which some crystalline CaP forms at pH 8 to pH 9 and ACP forms at pH 10 to pH 11 [23]. The oral environment, on the whole, is composed of saliva and gingival fluid, which are rich with calcium ions, phosphate ions, and fluoride ions. In a neutral pH, these components bring a dynamic equilibrium between the HAP in the teeth and in the oral fluid [26,27]. When the pH decreases to acidic (pH below 5.5), the oral environment becomes undersaturated, causing HAP crystals to release from the teeth into the oral biofilm, resulting in mineral dissolution. In conditions where the pH increases to over 4.5, the increased release of PO 4 3− and Ca 2+ ions causes supersaturation in the oral fluid. This process leads to the precipitation of the minerals back to the enamel surface [28,29].
Four commercial toothpastes were tested to evaluate the effect of free calcium release after being combined with TCP powder and TCP/ACP powder. We observed that the free calcium concentration increased significantly no matter which brand of toothpaste was combined with the 10% or 25% TCP powder. The concentration of free calcium increased even more in toothpaste containing the mixed TCP/ACP powder. It was previously believed that ACP could be converted to α-TCP and β-TCP through the sintering process [24,25]. Some previously published studies discovered that ACP has a potential role in promoting the HAP assembly into highly-ordered structures [30,31]. High-order HA architectures were detected when the initial nanosphere particles were aggregated, with the structure consisting of ACP shells and HA cores. Similarly, Posner et al. [32] demonstrated that ACP contains a well-defined local structural unit with a compositional constancy covering the broad conditions of preparative solutions, indicating the presence of a core structure. Based on previous findings, we presume that a core-shell structure might have been formed during the sintering process in the present study. As illustrated in Figure 6, the proposed mechanism is one in which the formed core-shell structure contained an inner ACP core coated with an outer TCP shell. The inner ACP core released free calcium at the same time as the outer TCP shell released free calcium. Through this mechanism, the toothpaste containing TCP/ACP powder released a higher concentration of free calcium than the toothpaste containing TCP alone, which could prove beneficial in promoting enamel surface remineralization. However, the possibility of increased formation of calculus should be evaluated due to more free calcium being released into the oral environment. Excess calcium can be retained easily in the oral cavity. Calculus may occur when calcium is deposited gradually [33]. The most direct preventive method would be to add anti-calculus agents to the toothpaste [34]. As long as sufficient anti-calculus agents are included in the toothpaste, the recurrence rate of calculus can be significantly reduced [34][35][36]. Experimentally, the toothpaste containing mixed β-TCP/ACP (2:1) powder is beneficial for the remineralization of enamel, but it can lead to other environmental changes in the oral cavity. These issues need to be further investigated in the future. Figure 6. The proposed mechanism of free calcium release from core-shell-structured ACP.

Conclusions
The present study fully characterized β-TCP and ACP powders fabricated using a twostage sintering process. The β-TCP/ACP powder mixed in a 2:1 ratio could stably release a free calcium concentration into water with a pH range of 5 to 9. Adding β-TCP/ACP mixed powder to toothpaste could release nearly twice as many free calcium ions than adding β-TCP powder alone. Thus, the high calcium concentration released from toothpastes containing β-TCP/ACP mixed powder might be beneficial as an effective remineralization agent when applied in dental care.