Evaluation of Two Different Types of Mineral Trioxide Aggregate Cements as Direct Pulp Capping Agents in Human Teeth

: Traumatic human dental injuries involving the pulp might necessitate direct capping procedures. This clinical study aimed to analyse the histological outcomes using two different direct capping materials. Twenty patients with bilateral premolars, scheduled for orthodontic extraction, were selected. The teeth were treated either using ProRoot MTA or RetroMTA. All patients were recalled after 30 and 60 days for teeth extraction. The histopathologically stained specimens were blindly evaluated using hard tissue bridge formation, inﬂammatory reaction and pulpal ﬁndings criteria. Data were evaluated statistically. Results: After 60 days, only the parameter for hard tissue bridge formation showed signiﬁcant difference in the ProRoot MTA group ( p = 0.010), while both direct capping materials performed similarly regarding inﬂammatory pulp reaction and pulpal ﬁndings. Although, during the ﬁrst 30 days, RetroMTA presented better results in terms of continuity, morphology, hard tissue bridge localisation, and extension/general state of the inﬂammatory reaction, the continuity was better at 60 days when ProRoot MTA was applied. Treatment with RetroMTA healed the pulpal tissue faster compared with ProRoot MTA but it seemed to be rather a reparative process.


Introduction
Dental trauma is a significant problem in young people. It has been reported that most dental traumatic injuries involve anterior teeth [1]. These traumatic injuries can cause irreparable damage to the young permanent teeth, not only at the time of accident, but also during the post-treatment period. Maintaining the vitality of the dentin-pulp complex is a high priority in cases of traumatic pulp exposures, since vital functioning pulp is capable of initiating several defence mechanisms to protect the body from bacterial invasion. Once pulpal tissue is exposed to the oral environment, among many other processes, repair or regeneration might occur, which is achieved by encouraging the formation of a "dentin bridge", using direct pulp capping (DPC) procedures. These involve the medical or addressing material application upon exposed tissue. Several prior studies indicated calcium hydroxide as gold standard for pulp capping [2][3][4]. However, performed studies have also reported disadvantages using calcium hydroxide such as dentin barrier tunnels, obliteration of the pulp chamber caused by extensive dentin formation, high oral fluid solubility, and a lack of degradation and adhesion after acid etching [5][6][7][8]. Mineral trioxide aggregate is one commonly used pulp capping agent, which was first evaluated by Pitt Ford et al. in monkey's teeth, showing a superior performance of MTA compared with calcium hydroxide [9].
MTA has several advantages over calcium hydroxide, such as lower solubility, improved mechanical strength, better marginal adaptation, and sealing ability [10]. However, ProRoot MTA has disadvantages such as long setting time and causes discoloration of teeth [11,12]. Hence, several MTA-like materials have been developed with improved physical properties. Recently, RetroMTA (BioMTA, Seoul, Korea), consisting of a hydraulic calcium zirconia complex, was introduced for pulp capping, pulpotomy, perforation repair, and as a base material [13,14]. The complex is a mixture of calcium carbonate, aluminium oxide, silicone dioxide, and zirconium oxide.
In RetroMTA, similar components are present as in ProRoot MTA but less heavy metal contents than ProRoot MTA (Table 1). Also, zirconium oxide has been added as an alternative radiopacifier to bismuth oxide to prevent discoloration [15]. It has been stated that RetroMTA has good biocompatibility, strong antibacterial effect, good sealing ability, fast setting (2.5 min) and no wash out. Chung C.J. et al. reported RetroMTA to have similar effects as ProRoot MTA on human pulp cells and can be used as pulp capping material. Only few clinial studies compared the efficacy of RetroMTA and ProRoot MTA as direct pulp capping material. Therefore, the aim of this clinical trial was to compare the efficacy of RetroMTA and ProRoot MTA in human dental pulp tissue healing when used as direct capping agent. The null hypothesis was there is no significant difference in hard tissue formation and inflammatory pulp response with RetroMTA and ProRoot MTA when evaluated after 30 or 60 days of application.

Inclusion and Exclusion Criteria
Twenty patients (ages 18-30) having contra lateral premolar teeth, scheduled for extraction because of orthodontic reasons were selected. A total of 40 teeth, 2 in each patient were selected. A split mouth design was used, where both MTA materials were applied in the same patient. Sample size was calculated with 95% confidence level and 80% power, according to existing literature [16]. Patients suffering from any systemic disease were excluded. The exclusion criteria were teeth with decay, restorations, trauma, periapical lesions, and periodontal pathologies. If teeth could not be isolated using a rubber dam and if patients were allergic to local anaesthesia, with systemic diseases, or when extraction is contraindicated, were also excluded. Informed consent of each patient was a prerequisite for participation in the study.

Clinical Procedure
Ethical clearance was approved from the institutional review board (IEC 859/2016). This study was registered in Clinical Trial Registry of India (CTRI/2017/10/010303) and carried out at the Department of Conservative Dentistry & Endodontics, Manipal College of Dental Sciences, Manipal, Karnataka, India. Following a detailed explanation of the experimental study purpose, clinical procedures, and possible risks, informed consent was obtained from each patient. Absence of decay, restorations, trauma, periapical lesions, and periodontal pathologies was assured throughout clinical and radiographical examination of all teeth. Teeth which could not be isolated under a rubber dam were excluded. The pulp sensibility of all the experimental teeth was tested using thermal (cold test, Endo-Frost, Coltene, Cuyahoga Falls, OH, USA) and electric stimuli (Parkell Electronics Division, Farmingdale, NY, USA). In each patient, local anaesthesia (Septodont, Saint-Maur-des-Fosses, France) was performed, followed by rubber dam isolation, and cleansing of the surrounding regions (clamp, dam, and tooth) using a 0.2% chlorhexidine gluconate solution. High speed diamond burs (Horico Dental, Berlin, Germany) under water coolant were used to prepare class I occlusal standardized cavities with an occlusal depth of 3 ± 0.2 mm, mesiodistal width of 4 ± 0.5 mm, and buccolingual width of 3 ± 0.2 mm. After exposure of the pulp in the cavity centre on the pulpal floor using a sterilized round bur under water cooling, haemostasis was obtained by application of gentle pressure using a saline solution moistened cotton pellet. The tooth was then randomly treated (coin toss method) with either ProRoot MTA (Dentsply Sirona) or RetroMTA (BioMTA). The same procedure was applied on the contra lateral premolar. Both MTA cements were prepared and applied according to manufacturer's instructions. After application of the pulp capping material, a thin layer or resin-modified glass ionomer cement (Ionolux, VOCO GmbH, Cuxhaven, Germany) was placed over the tested capping materials as a liner and the access hole was restored using a universal resin composite (Filtek Z350 XT, 3M ESPE, St. Paul, MN, USA). All the procedures were carried out by a single operator, experienced in performing vital pulp therapy. All patients were recalled after 30 and 60 days for teeth extraction and histopathological analysis. In this period, if the patient experienced any pain in the restored test with the test agents, it was extracted immediately.

Tissue Processing for Microscopic Examination
A total of 10 teeth from each group were extracted after 30 days, and 10 further teeth were extracted after 60 days, for histopathological analysis. Presence and/or absence of post-operative tenderness to percussion, sensitivity, and pain were evaluation by questionnaires of the patients. Furthermore, Pulp sensibility tests were repeated according to the preoperatively performed ones at the time points 30 and 60 days. Oral surgeons extracted the teeth atraumatically, and orthodontists continued the orthodontic treatment. Following extraction, the apical 3 mm of the roots were sectioned to enable penetration of the 10% buffered formalin solution for 72 h. After blind coding for histomorphological processing and examination, the specimens were decalcified using 50% formic acid-sodium citrate for a 6 to 8 weeks period and prepared and embedded in paraffin. Between 10 and 12 sections, 4 or 5 6 micrometre sections were obtained by cutting using a microtome in a parallel direction to the vertical axis of the tooth. Sections were then mounted on glass slides, stained with haematoxylin-eosin, blindly evaluated by an experienced and calibrated oral and maxillofacial pathologist, according to the criteria in Tables 2-4. Each histomorphological event was evaluated with a 1-4 score system, with 1 being the best and 4 the worst result [17].

Statistical Analysis
Statistical analysis was performed with R Project for Statistical Computing 3.3.0 and Microsoft Excel 2013. The normality of the data distribution and the homogeneity of group variances were verified by the Kolmogorov-Smirnov test. Chi square test was performed for the comparison between the Proroot MTA and RetroMTA. p < 0.05 was considered as significant.

Results
All selected patients and treated teeth could be evaluated. The score percentages for each group in each criterion are shown after 30 days in Table 5, and after 60 days in Table 6. Considering hard tissue bridge formation after 30 days, significant differences could be found when ProRoot and RetroMTA were compared. As for the parameters-continuity (p = 0.029), morphology (p = 0.023), and localisation (p < 0.001)-the RetroMTA performed significantly better compared with ProRoot MTA.
After 60 days, only continuity, the hard tissue bridge formation parameter, showed significant difference, in favour of the direct capping material ProRoot MTA (p = 0.010) (Figure 1). Considering hard tissue bridge formation after 30 days, significant differences could be found when ProRoot and RetroMTA were compared. As for the parameterscontinuity (p = 0.029), morphology (p = 0.023), and localisation (p < 0.001)-the RetroMTA performed significantly better compared with ProRoot MTA.
After 60 days, only continuity, the hard tissue bridge formation parameter, showed significant difference, in favour of the direct capping material ProRoot MTA (p = 0.010) (Figure 1). The pulpal inflammatory response with the criteria (intensity, extension, and general state) no significance could be observed 30 days after the treatment for the parameter intensity (p = 1.00), while RetroMTA performed better in terms of extension (p = 0.007) and general state (p = 0.033).
Pulpal findings including the parameters (giant cells and particles) performed only significantly different for the parameter particles when both direct capping materials were compared after 30 days, in favour of RetroMTA (p = 0.048).
After 60 days, both direct capping materials Pro Root and RetroMTA performed similar regarding inflammatory response of the pulp and pulpal findings.

Discussion
This clinical study was conducted in teeth with a healthy uninflamed pulp using two different capping materials ProRoot MTA and RetroMTA, which are hydraulic biocompatible cements [18] with similar biological effects [19]. The null hypothesis was rejected for the parameters of hard tissue formation, inflammatory pulp response, and pulpal findings, except for intensity and giant cells number after 30 days, and for continuity after 60.
As for the hard tissue bridge, the RetroMTA results of this study showed better results regarding the parameters continuity, morphology, and localisation, while ProRoot MTA performed better regarding the parameter thickness. However, after 60 days, the only significant parameter was continuity, in favour of ProRoot MTA. The complete bridge formation is expected to take place 60-90 days after pulp capping [20,21].
The findings of this study are in accordance with one histological study, which evaluated direct pulp capping performance in dog's teeth, where no significance could be found between the two test pulp capping materials RetroMTA and ProRoot MTA, where a hard tissue bridge was formed in direct contact to the vital pulp [18]. Kang et al. also sustained high success rate up to 1 year, with no significant differences between outcomes treated with ProRoot MTA (96.0%) and RetroMTA(96.0%) [22].
The dentine bridge, in the form of a calcified barrier, is usually considered a favourable response to capping therapy. In conjunction to vital signs, it supports that the pulp condition is healthy, leading to physiologic dentin deposition. Mass, E. et al. reported that a calcified barrier was found in almost 70% of teeth [23], compared with 55%-64% in previous studies [24,25].
The thickness of the dentine bridge in the 60 days sample of RetroMTA is less than 30 days. It is proof that the thickness of the dentinal bridge may vary depending on the location and angle of sectioning; therefore, it is more critical to evaluate the condition of pulp beside the bridge thickness, continuity, and quality, while assessing the success of the direct pulp capping material. The initiation is induced throughout initiation of reparative dentinogenesis in the case of MTA [26,27]. Pulp cells are aligned in proximity to crystals, built in a homogenous zone along the pulp-MTA interface. After MTA hardening, calcium oxide is formed, which can react with tissue fluids to produce calcium hydroxide [28]. After contact with pulp tissue, MTA presents some structures that are similar to calcite crystals, which attract fibronectin, which is generally responsible for cellular adhesion and differentiation [29]. Some studies found necrotic tissue next to the hard tissue bridge, which is assumed to be caused by MTA throughout coagulation in contact with pulp connective tissue [26]. The randomized controlled trial, conducted by Kang et al. [23], using three different MTA materials (ProRoot, Ortho, and RetroMTA), stated that the partial pulpotomy performed in permanent teeth showed favourable results, while clinical and radiographic results showed no difference after one year. In contradiction, one study found significantly worse results after 8 weeks using RetroMTA concerning pulp morphology and hard tissue bridge thickness compared to ProRoot. The hard tissue was described as a tubular reparative dentine. However, one study reported a lack of mineralized tissue and rather regular dentin with parallel tubules and rather amorphous and irregularly calcified tissue with lacunae of necrotic debris [23]. This finding may explain the changes after 60 days, and the less favourable performance of RetroMTA after 60 days. The quality of the hard tissue bridge relates to the pulpal health state after application of MTA and the pulp capping procedure [30,31].
For tooth survival, the pulp status and inflammatory response after pulp capping material application is more important compared to the thickness of the dentine bridge. The inflammatory response of the pulp with the criteria (intensity, extension, and general state) showed no significance between both tested cements after 60 days. RetroMTA is a hard-setting hydrophilic cement consisting of fine, hydrophilic particles that set in the presence of moisture and form a hard barrier. In contrast to Ca(OH) 2 suspension, calcium silicate cements are not resorbable; therefore, they provide long-term protection against the invasion of microorganisms [32,33]. The quality of the hard tissue influences the state of pulpal health after direct pulp capping [30,31]. Furthermore, the formation of a hard tissue bridge does not mean that the pulp tissue will be sealed completely, because the hard tissue is permeable initially [34]. The formation of a mineralized barrier after capping with RetroMTA was reported as unpredictable, as it was incomplete and showed channels, which can be supported by the findings of this study as initial better results after 30 days compared to ProRoot MTA disappear, while the values for ProRoot MTA remain stable. The inflammation intensity and extension of RetroMTA teeth increased, while the general state of the pulp decreased.
No significant difference regarding the presence of microorganisms was found after 60 days. This means that, although the MTA particles dissolved faster in the RetroMTA group, the bacteriostatic action of both MTAs, per se, was similar, and was enough to reduce the number of viable bacteria near the pulp exposure.
Study limitations were the healthy uninflamed teeth, which don't present the response that occurs in a clinical setting of an inflamed pulp, as the pathological changes of inflammation, odontoblasts absence, and tertiary dentine formation are missing. As for the bacteria detection, low sensibility of the histochemical staining technique for the detection of bacteria makes their identification difficult, mainly when there is a small number of such microorganisms [35]. Moreover, bacteria are easily removed from dental tissue during histologic preparation [36,37]. Although the use of healthy teeth allows standardisation, the effects of ProMTA and RetroMTA should also be evaluated in a clinical setting using infected and inflamed teeth.

Conclusions
The results of the present study indicated that RetroMTA might be a valuable ProRoot MTA alternative, regarding the tested parameters pulpal findings, including inflammatory response and hard tissue bridge formation. Although, during the first 30 days period, RetroMTA presented better results in terms of continuity, localisation, and morphology of the formed hard tissue bridge and extension and inflammatory pulp response, the continuity improved after 60 days when ProRoot MTA was applied.
MTA seemed to heal the pulp tissue at a faster rate for RetroMTA compared to ProRoot MTA. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Conflicts of Interest:
The authors declare no conflict of interest.