The success of pulp capping is dependent on the preservation of vital pulp tissue and the formation of tertiary dentin
[23, 24]. For this purpose, MTA has been in widespread use clinically. However, MTA does not have good handling properties when prepared according to the manufacturers’ instructions, and the setting time is relatively long after mixing
[20, 25]. Meanwhile, CP cements have been interesting as a pulp capping agent, because of favorable biocompatibility and osteogenic/odontogenic potentials
[13, 26, 27]. However, due to some drawbacks such as limited antibacterial properties, long setting time, and compressive strength, the application of CP cement for vital pulp therapy is limited
[28, 29]. Recently, fast-setting α-TCP cement was experimentally developed both for bone repair procedures and vital pulp therapy to overcome one of the physical disadvantages of conventional CP cements. Kurashina demonstrated that α-TCP-based cement is also a promising material as a bone substitute
. In fact, the β-type is considered a more popular TCP variant for bone repair, but the α-type offers more resistance to degradation by tissue
[30, 31]. This characteristic of α-type TCP would be more appropriate for vital pulp therapy. In fact, the present study is not the first study which attempted to show the potential clinical application of fast-setting CP cements. Miyamoto et al. reported that fast-setting CP cements may be used in a wide range of clinical fields, such as oral and maxillofacial surgery
. However, their study only investigated the setting behavior of the calcium phosphate cement, and did not investigate biological effects. In other words, there has been no study to determine whether the fast-setting α-TCP possesses odontogenic activity and can induce tertiary dentin formation, the ultimate goal of pulp capping. Therefore, we investigated its physical and biological/odontogenic effects in comparison with the currently used material, MTA.
First, we evaluated the physical properties of α-TCP including setting time, pH, solubility, and compressive strength in comparison with MTA. The setting time of α-TCP was significantly shorter than that of MTA (P < 0.05). α-TCP consists of small particles of CP. It is generally believed that the use of small particles increases the surface contact of the particles with the mixing liquid, which provides rapid setting and ease of handling. Due to this property, α-TCP might be used in a single-visit scenario without additional required appointment. On the contrary, the solubility of α-TCP was significantly higher than that of MTA (P < 0.05). This result was anticipated because CP cement, as a bone repair material, is essentially designed to be degraded and substituted by bone. However, this property can be considered negatively for pulp capping procedures. Furthermore, the compressive strength of α-TCP was significantly lower than that of MTA (P < 0.05). The ISO standard in terms of measuring compressive strength for a pulp capping material has not been developed. Therefore, ISO 3107:2004 was selected as a guideline for evaluation of the material properties. It is traditionally recommended that fillers be strong enough to resist the stress which is applied through an amalgam condensation
. Lately, however, tooth-colored, non-pressure-generated materials have been widely used instead of amalgam. In this respect, the importance of compressive strength is reduced for pulp capping materials.
Next, we investigated the biocompatibility of the two materials by evaluating the effects of the tested materials on cell morphology and viability. It is considered favorable for a pulp capping material to be biocompatible, because the material is then less likely to induce a response such as pulpal inflammation
. In our study, MTA and α-TCP had similar effects on cell viability shown by the MTT assay until day 7 (P > 0.05). On day 14, however, α-TCP showed higher cell viability compared to ProRoot (P < 0.05) (Figure
1D). Furthermore, SEM observations revealed that hDPCs cultured directly on MTA or α-TCP for 3 days appeared to be flat and exhibited well-defined cytoplasmic extensions (Figure
1C and D). Our study is supported by previous studies on the biocompatibility of CP cements
[35, 36], and indicates that the biocompatibility of fast-setting CP cement is comparable to that of MTA.
We also investigated whether α-TCP facilitated odontoblastic differentiation of hDPCs in comparison with MTA. MTA is considered to facilitate odontoblastic differentiation of hDPCs
[37–40]. In addition, several studies have shown that CP cement has a similar mineralization ability compared to MTA
[35, 36, 41]. In the present study, we showed that MTA and α-TCP promoted odontoblastic differentiation to a similar degree, as evidenced by the formation of mineralization nodules, and the expression of odontogenic-related markers. As shown in Figure
3, notably, the relative quantities of odontogenic-related markers such as DSPP, DMP1, and ON proteins were significantly higher in MTA- and α-TCP-treated cells compared to the medium only-treated cells of the control group (P < 0.05). However, there was no significant difference between the two experimental groups (P > 0.05). In immunofluorescence analyses, we observed that the signals in MTA- and α-TCP-treated cells were stronger compared to the cells of the control group (Figure
4). It is suggested that the ions released from MTA or CP cement, such as Ca, P, and Si, promote differentiation and mineralization of the cells through ion-mediated reactions
[40, 42, 43]. EDS analysis of the current study revealed that MTA and α-TCP contain these elements, which might affect the expression of the mineralization phenotypes in vitro (Figure
2C and D). Overall, these results indicate that α-TCP possesses a similar ability with MTA in terms of promoting odontogenic potential of hDPCs.
Lastly, we investigated whether α-TCP induces the formation of tertiary dentin in vivo. Similar to in vitro results, there was no difference between MTA and α-TCP in terms of tertiary dentin formation (P > 0.05). Tertiary dentin with complete continuity was formed directly underneath the capping materials and the pulp exposure area in all samples of the two tested groups (Table
2 and Figure
5). There have been several in vivo studies indicating that CP-based cement induces the formation of tertiary dentin in direct contact with the material, and revealing its potential for use as a pulp capping agent
[9, 10, 13, 44]. The alkaline environment caused by a material in the pulp space is required for reparative dentin formation, because the alkalinity appears to result in mild stimulation of cell differentiation
[45, 46]. In the present study, both MTA and α-TCP had alkaline pH values that remained consistently high for 14 days, although the pH values of MTA were significantly higher than those of α-TCP (Figure
1A). Similar to our result, Tagaya et al. reported that the pH of the CP-based cement solution never exceed 8.0, even after about one month of storage, and this provides a mildly alkaline environment for pulpotomy
. Along with ionic release or other possible mechanisms, this alkaline environment might promote reparative dentin formation in vivo.