Introduction

Mineral trioxide aggregate (MTA) is a repair material composed of fine hydrophilic particles of tri/dicalcium silicate, tricalcium aluminate, tricalcium oxide, and silicate oxide [1]. MTA has been initially indicated for sealing root perforations and as a root-end filling material, improving treatment outcomes due to its sealing ability, biocompatibility, bioactivity, and antimicrobial effect [1,2,3]. Its properties have encouraged the use of MTA in many other clinical applications. MTA induces satisfactory outcomes, even in cases when the material might get in contact with pulp tissue, fluids, or blood [4, 5]. However, the long setting time, difficult handling, and possibility of tooth discoloration are some drawbacks of this material [1,2,3, 6, 7].

Nowadays, two commercially available materials that still maintain the MTA original formulation are ProRoot MTA (Dentsply Sirona, York, PA, USA) and MTA Angelus (MTA-A; Angelus Soluções Odontologicas, Londrina, PR, Brazil). Several other calcium silicate-based cements have been developed aiming to optimize the clinical performance. Among them, Biodentine (BD; Septodont, Saint-Maur-der-Fósses, France) and NeoMTA Plus (Avalon Biomed Inc., Brandenton, FL, USA) are two contemporary bioceramic cements. BD consists of a powder component containing di/tricalcium silicate, calcium carbonate and oxide filler, iron oxide, and a radiopacifier (zirconium oxide). The liquid component of BD contains calcium chloride as an accelerator and a hydrosoluble polymer that serves as a water-reducing agent [8]. BD presents adequate properties, such as low solubility, high calcium release, short setting time, radiopacity [9, 10], low cytotoxicity, and appropriate response when in contact with connective tissues [11,12,13].

MTA Plus (Avalon Biomed Inc., Bradenton, FL, USA) presents similar composition to the original MTA, but it is ground finer and provided with either a gel or water ampoule for mixing. Recently, MTA Plus was replaced by NeoMTA Plus, which maintains the main components of the previous formulation, except for the addition of tantalum oxide instead of bismuth oxide as radiopacifier [14]. NeoMTA Plus has shown satisfactory sealing ability, resistance to leakage [15], adequate radiopacity, and ability to release calcium and hydroxyl ions [16]. A chemical characterization of NeoMTA Plus and BD concluded that these materials were suitable to be used in the treatment of immature teeth because they produced calcium hydroxide, which is necessary to induce dentin bridge formation and continued root formation [15]. Additionally, they can be used as an alternative to MTA Plus, as they do not result in discoloration, due to the absence of bismuth oxide in both materials [14].

The literature has provided evidence that points BD and MTA-A as materials with appropriate physicochemical and biological properties [2, 3, 8,9,10, 13, 17, 18]. However, there is insufficient information on NeoMTA Plus, particularly in regard to its biological properties, such as biocompatibility and osteoinductive potential [13, 19].

Therefore, the aim of the present study was to compare the bone tissue reaction, setting time, solubility, and pH of NeoMTA Plus, Biodentine (BD), and MTA Angelus (MTA-A). The hypothesis tested was that the use of NeoMTA Plus or BD would improve the bone tissue repair, and they would present shorter setting time, lower solubility, and higher pH compared to MTA-A because of the physico-chemical enhancements that the contemporary materials have undergo.

Materials and methods

This study was approved by the local research ethics committee (protocol no. 30488). NeoMTA Plus, BD, and MTA-A were evaluated in relation to setting time, solubility, pH, and bone tissue reaction. All materials were mixed according to the manufacturers’ instructions. NeoMTA Plus can be mixed from 1:1 to 4:1 depending on the needed consistency. Herein, a 1:1 proportion was used.

Setting time

The initial and final setting times were evaluated according to ASTM C266–15 [20]. Plaster molds (n = 7 for each material) with an internal diameter of 10 mm (± 0.1 mm) and a height of 2 mm (± 0.1 mm) were filled with the mixed material. Samples were maintained for 5 min in an incubator at 37 °C and 99 ± 5% relative humidity before initial setting time measurements. Initial setting was measured with a Gilmore-type needle [diameter of 2 mm (± 0.1 mm), a height of 5 mm, and a weight of 100 g (± 5 g)] that was carefully lowered onto the surface of the sample without exerting any further pressure. This procedure was repeated every 60 s until an impression was no longer visible on the material surface and, at this moment, the initial setting time was recorded. The final setting time (time elapsed from the beginning of mixing to the time at which no indentation was detected on the surface of the specimens) was determined with a bigger Gilmore needle [diameter of 1 mm (± 0.1 mm) and a weight of 456.5 g (± 5 g)].

Solubility

Solubility test was adapted from the American National Standard Institute/American Dental Association (ANSI/ADA) Specification No. 57, following previous studies [21, 22]. Samples of NeoMTA Plus (n = 11), BD (n = 11), and MTA-A (n = 11) were prepared using plastic molds with a height of 1.5 mm (± 0.1 mm) and an internal diameter of 7.75 mm (± 0.1 mm) [21]. The molds were placed on a glass plate and filled with the mixed material. Another plate was positioned over the samples to be stored in an incubator, at 37 °C and 95% relative humidity, for three times longer than the initial setting time of each material (provided by the manufacturers: 5, 12, and 10 min for NeoMTA Plus, BD, and MTA-A, respectively) [22]. The specimens were unmolded and weighed in a precision scale (Sartorius 1801MPS, Göttingen, Germany) to determine the initial mass. Following the initial weighing, the samples were immersed in 15-ml Falcon tubes (Cral artigos para laboratório Ltda., Cotia, Brazil) filled with 7.5 ml of deionized water [21] and maintained in the incubator (SX 1.4 DS, Sterilifer Ind. Com. Ltda., Diadema, Brazil) for 24 h. Subsequently, the specimens were removed from the incubator, slightly dried with absorbent papers, and placed in a drying chamber at 37 °C for 48 h. Then, samples were weighed in shifts of 24 h for 7 days. Samples were maintained in the incubator during all periods between weighing. Solubility was expressed by the percentage of material mass loss over time.

pH

For each type of material (n = 10), samples were prepared from polyethylene tubes with dimensions 10 mm in height by 1.6 mm in diameter. After the initial setting time (provided by the manufacturers), samples were inserted into 50-ml Falcon tubes (Cral artigos para laboratório Ltda.) containing 10 ml of deionized water (pH = 7.4). The measurements were taken using a pH meter (Digimed, Digicrom Analitica, Campo Grande, Brazil), at a constant temperature of 25 ± 2 °C, which was kept using an air conditioner, in the following periods: 1, 3, 12, and 24 h, and 7, 14, 21, and 28 days. Samples were maintained in at 37 °C in an incubator (SX 1.4 DS, Sterilifer Ind. Com. Ltda.) during all periods between measurements.

Bone tissue reaction

Bone tissue reactions to NeoMTA Plus and BD, compared to MTA-A, were evaluated in femurs of 48 Wistar rats after 3 experimental periods (7, 30, and 90 days). Two experimental sets were performed: NeoMTA vs. MTA-A (n = 8 per period) and BD vs. MTA-A (n = 8 per period). Control groups were included in both experimental sets (n = 8 per period). Surgical procedures were conducted similarly as previously described [23]. The animals were anesthetized with 0.008 ml/100 g ketamine (Virbac do Brasil Industria e Comércio Ltda., São Paulo, Brazil) and 0.004 ml/100 g 2% xylazine hydrochloride (Virbac do Brasil Industria e Comércio Ltda.). Then, trichotomy was performed on the right leg and a 4-cm-long incision was made on the skin with a scalpel (Blade #15, Ethicon; Johnson & Johnson, São José dos Campos, Brazil), tissues were separated by layers, and the periosteum was incised. For each rat, a new low-speed sterile trephine no. 4 (KG Sorensen, Cotia, Brazil) was used under constant irrigation with saline and aspiration, positioned perpendicularly to the cortical femur surface, until bone disruption was verified. Three equidistant 4-mm-diameter cavities were prepared. In 24 animals, surgical cavities were randomly designated, in each rat, to NeoMTA Plus, MTA-A, and control (empty cavity) groups; in the remaining 24 animals, cavities were also randomly distributed in BD, MTA-A, and control groups.

The amount of 0.2 ml of cement was introduced into the designated cavity using insulin syringes (Injex, Ourinhos, Brazil). After, the material was accommodated and smoothed with a damp cotton pellet. The wound was sutured in layers (Vicryl Ethicon, Johnson & Johnson, New Brunswick, NJ, USA). After experimental procedures, 50 mg/kg opioid analgesic (Tramal 50, Pfizer, New York, NY, USA) was injected intramuscularly.

By the end of each experimental period, 16 animals (8 from each experimental set) were randomly euthanized using intraperitoneal thiopental 120 mg/kg in mixed solution with anesthetic (lidocaine 10 mg/ml). The femur was isolated and transversely sectioned with a low-speed diamond disk (7020, KG Sorensen, Cotia, Brazil) to separate the surgical cavities. Each fragment was individually stored in 10% neutral-buffered formalin for 48 h. After decalcification with 5% nitric acid, the samples were set in paraffin blocks and processed for histologic analysis. Sections with 3-μm thickness were cut transversely to the femur long axis, mounted on slides (Kasvi, São José dos Pinhais, Brazil), and stained with hematoxylin-eosin (Harris Hematoxylin, Dinâmica Química Contemporânea Ltda., Diadema, Brazil; 1% eosin, Êxodo Científica, Hortolândia, Brazil). Slices were analyzed on a light microscope (Model Lambda LQT 2; ATTO Instruments Co, Hong Kong, China) using ×40, ×100, ×300, and ×400 magnification.

One blinded and calibrated examiner (k = 0.748, P < 0.001) evaluated the histological characteristics of repair as follows: (1) partial closure of the bone defect area by large amount of fibrous connective tissue and few areas of mineralized tissue; (2) partial closure of the bone defect area by mineralized tissue with interspersed fibrous connective tissue (trabecular bone); (3) complete closure of the bone defect area by trabecular bone; (4) complete closure of the bone defect area by large amount of mineralized tissue with several incremental lines (lamellar bone) and few areas of trabecular bone; and (5) complete closure of the defect by lamellar bone, demonstrating complete regeneration.

Statistical analysis

Statistical analysis was performed with GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA) (α = 0.05). Initial and final setting times were compared between groups by one-way ANOVA and Tukey’s test. Solubility and pH data were submitted to intragroup comparisons (between periods) using RM-ANOVA and Tukey’s test, and the comparison between groups (in each period) was analyzed by two-way ANOVA and Tukey’s test. Data from bone tissue repair had intergroup comparisons performed by Friedman’s test, and the three experimental periods in each group were compared by Kruskal-Wallis test followed by the Dunn’s test.

Results

Setting time, solubility, and pH results are shown in Table 1. NeoMTA Plus had the longest initial and final setting times followed by MTA-A and BD (P < 0.05). The tested materials had mass loss over time, except MTA-A that had an increase in mass at 24 h (3.53%). During all experimental period, the mass loss showed by BD was significantly higher than MTA-A, and both cements were similar to NeoMTA Plus. Also, all materials showed an alkaline pH. BD presented higher pH in most of the periods assessed when compared to the other two materials (P < 0.05), and it was able to maintain the pH above 10 throughout the experiment. NeoMTA showed similar pH values when compared to the MTA-A in the most of the periods assessed (P > 0.05), except in 12 and 24 h (P < 0.05). Overall, for all types of cements tested, the pH means were highest from 12 h to 7 days.

Table 1 Mean and standard deviation (± SD) of initial and final setting times (min), solubility expressed as mass loss (%), in relation to initial mass (g) and pH values measured in different experimental periods of NeoMTA, BD, and MTA-A
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No inflammatory infiltrate was verified during the whole experiment. Figures 1 and 2 summarize score comparisons for bone repair between groups and periods (for each experimental set). No significant difference was found between cements and control group in any experimental period. All groups, in both sets, showed a better bone repair at 90 than at the 7 days (P < 0.05). Moreover, on the NeoMTA Plus vs. MTA-A experimental set, all groups had an improvement of the bone repair from 7 to 30 days (P < 0.05). Figures 3 and 4 illustrate the scores attributed to the different groups in each experimental period.

Fig. 1
figure 1

Box plot graph representation of bone repair scores observed for NeoMTA Plus, MTA-A, and control groups in all experimental periods. Horizontal bars indicate significant differences between periods in each group (P < 0.05)

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Fig. 2
figure 2

Box plot graph representation of bone repair scores observed for BD, MTA-A, and control groups in all experimental periods. Horizontal bars indicate significant differences between periods in each group (P < 0.05)

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Fig. 3
figure 3

Histologic features with ×100 magnification of NeoMTA Plus, MTA-A, and control in each period. a Score 2. b Score 4. c Score 5. d Score 1. e Score 4. f Score 5. g Score 3. h Score 3. i Score 5. Arrows indicate the bone defect areas. BM indicates bone marrow area. Magnification bar = 100 μm

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Fig. 4
figure 4

Histologic features with ×100 magnification of BD, MTA-A, and control in each period. a Score 2. b Score 4. c Score 5. d Score 3. e Score 3. f Score 5. g Score 3. h Score 5. i Score 4. Arrows indicate the bone defect areas. BM indicates bone marrow area. Magnification bar = 100 μm

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Discussion

This study evaluated the setting time, solubility, pH, and bone response to NeoMTA Plus, BD, and MTA-A. BD distinguished itself from the other two materials in setting time, solubility, and pH analysis. Regarding bone tissue reaction, both BD and NeoMTA Plus exhibited similar outcomes compared to MTA-A.

BD presented the shortest setting time among the tested materials. BD faster setting, in comparison to the original MTA, is achieved by increasing particle size, by adding calcium chloride to the liquid component, and by decreasing the liquid content [8]. In contrast, NeoMTA Plus had the highest initial and final setting times, in agreement with a previous study [16]. According to those authors, even when using a 1:1 powder-gel proportion, the final setting time (69.98 min) was lower than the time informed by the manufacturer for this consistency (5 h) [16, 24]. Therefore, taking into consideration the three following factors: (i) the possibility that the material will be able to gain resistance within 5 min after mixing [24], (ii) the appropriate handling using 1:1 proportion, and (iii) the final setting time observed herein (67.98 ± 0.28 min), it is possible to affirm that NeoMTA Plus can be used in clinical procedures performed in a single appointment.

It was expected that a shorter setting time would reduce the solubility of the material, considering the contact of the calcium silicate-based cements with body fluids [25, 26]. Although BD showed the shortest setting times, it presented the highest solubility over time. Taking into consideration the ANSI/ADA Specification No. 57, BD had solubility higher than 3%, which is not the ideal for this kind of cement in a 24-h period. The modification in the ANSI/ADA size and shape of the specimens performed in this study might have increased the mass loss, which also was observed previously [27]. Differently, an investigation using samples according to the ISO 6876:2001 recommendations showed a lower solubility of BD in double-distillated water in 24 h [28]. NeoMTA Plus and MTA-A performed in accordance with the ANSI/ADA Specification No. 57 (< 3%). While NeoMTA Plus had a mean mass loss equal to 2.18% at 24 h, MTA-A showed an increased mean mass of 3.53%. This result may be associated to the hydration mechanism of mineral trioxide aggregate and the setting expansion (formation of calcium silicate hydrate gel C-S-H soon after mixing powder and water are combined) and the formation of needlelike crystals [29, 30].

Previous studies involving MTA Plus and ProRoot MTA showed that specimens had high solubility values when shorter periods of setting (lower than 24 h) were used before samples’ immersion in water [31, 32]. In the present investigation, specimens set for a period three times higher than the initial setting time. These short periods also might have induced considerable mass loss. Nevertheless, solubility might be lower in vivo than in vitro due to contact with biological fluids and the formation of a calcium phosphate layer [23,24,25].

A lower solubility for calcium silicate-based cements is desirable to provide a long-term seal and avoid leakage [33]. Moreover, OH− and Ca2+ ion release, as result of cement hydration, is related to the solubility; it also generates an alkaline environment and stimulates healing [34]. All groups showed alkaline pH, with highest values between 12 h and 7 days. These results suggest that all investigated materials are suitable to be used in acidic environments, since a high pH is associated with antimicrobial properties, including efficacy against Enterococcus faecalis [34]. Also, a high pH value may favor the tissue repair when calcium silicate-based cements are used [12].

Calcium silicate-based cements are expected to be in contact with body fluids. Thus, the deposit of materials directly in a bone cavity appears to be the best way to simulate a clinical situation [23, 35]. As expected for non-toxic materials, all groups allowed bone repair over time [23]. The absence of inflammatory infiltrate can be explained by the standardization of the surgical procedures. Intense and long-lasting inflammatory response, previously described by the literature [23, 35], may be result of material extrusion to bone marrow. The current investigation aimed to simulate placement without overfilling.

There is little information on the biological properties of NeoMTA Plus. This cement is similar to MTA Plus (Avalon Biomed Inc., Bradenton, FL, USA), differing only by the radiopacifier. Thus, favorable biological reactions to NeoMTA Plus, as those demonstrated for MTA Plus [11, 12], were expected. In a previous study, MTA Plus compared to MTA-A had higher alkaline phosphatase activity [36], an enzyme that plays an important role in mineral deposition [37]. Based on that, it is assumed that high phosphatase activity might have occurred in the surgical sites filled with NeoMTA Plus in the present investigation. This could influence the differences in bone reactions between NeoMTA Plus and MTA-A.

The rat tissue response to endodontic cement is an appropriate method to evaluate the material biological properties [38]. Considering that tricalcium silicate-based cements are indicated for sealing root perforations and as root-end filling materials [3], in a clinical situation, it is expected that the material stays in intimate contact with periodontal soft and hard tissues [39]. To simulate a clinical situation and to show the histological effects of materials in oral mucosa, the use of endodontic materials in direct contact with the animal tissue was proved to be better than the use of implants in polyethylene tubes [40]. Therefore, in this current study, the cements were inserted directly in the bone cavity [23].

Although the similar results regarding the tissue reaction found for the control and test groups might suggest that any of the tested materials could promote healing, it is important to highlight that in clinical situations (such as root perforation and root-end filling), it is essential to prevent infection for better prognosis [41]. Previous investigations showed that the high sealing ability of calcium silicate-based cements might contribute to prevent bacterial colonization [15, 42, 43].

Recently, the osteoinductive potential and bioactivity of BD and MTA Plus were tested in rabbit’s medullary bone [44]. In accordance with our study, the authors demonstrated good biocompatibility, slight inflammatory infiltrate, and no sign of necrosis at the interface with the pre-existing bone [43]. Moreover, MTA Plus exhibited bioactive potential and newly formed bone was observed next to BD [44]. In this present study, half of BD samples showed low initial biocompatibility (7 days), which could be justified by the early tissue inflammation, which was followed by tissue repair [12].

Conclusion

Based on the results and methods used, it can be concluded that all materials showed alkaline pH, appropriate setting time, and good biological responses in contact with rat’s bone tissue. Therefore, NeoMTA Plus, Biodentine, and MTA-Angelus were considered suitable to be used as root repair materials.