The effect of three additives on properties of mineral trioxide aggregate cements: a systematic review and meta-analysis of in vitro studies

Calcium silicate-based cements (CSCs) exhibit diverse applications in endodontics, encompassing procedures such as vital pulp treatments, apexification, perforation repair, and regenerative endodontic procedures [1]. ProRoot Mineral Trioxide Aggregate (ProRoot MTA; Dentsply, Tulsa, OK, USA) stands as the inaugural member of the CSCs family. Its introduction took place in 1993 comprising a composition of 75% Portland cement and 25% bismuth oxide [1]. Mineral Trioxide Aggregate (MTA) and other subsequently introduced CSCs demonstrate a diverse range of advantages, with numerous studies highlighting their notable feature of high biocompatibility [1, 2]. These materials undergo setting in the presence of moisture, yielding hydration products from the setting reaction, notably calcium silicate hydrate (CSH) gel and calcium hydroxide. The dissociation products emanating from calcium hydroxide contribute to alkalinity, and the presence of calcium ions offers an osteo/cemento inductive potential to the surrounding environment [3]. Consequently, CSCs not only demonstrate sufficient biocompatibility but also have the capability to promote the formation of a thin cementum-like layer in their vicinity. This phenomenon can be regarded as a biologic barrier, contributing to the improvement of their sealing ability [4]. In clinical applications, CSCs are positioned in direct proximity to pulpal and periodontal tissues, followed by the application of restorative materials with enhanced mechanical characteristics, such as composite resins. Consequently, while high compressive strength (CS) is not imperative for CSCs, their mechanical attributes should ensure sufficient compressive resistance against the subsequent restoration [3]. The extended setting time (ST) of CSCs, as a drawback, can impede the possibility of performing treatments in single-visit, which can therefore result in prolonged treatment time, need for scheduling further treatment sessions, and inter-appointment re-infection of the prepared root canal space [5]. According to the manufacturer's recommendation, the powder of MTA should be added to distilled water in a 3:1 ratio. This would result in a sandy and a bit gritty consistency and poor handling of the material [6].

Numerous efforts have been made to incorporate or eliminate diverse substances from the compositions of CSCs in order to enhance their properties [7]. For example, to enhance the handling characteristics, it has been suggested to add propylene glycol (PG) to the water used with MTA. Due to its higher viscosity and water solubility, a more clinically convenient consistency of MTA admixture will be provided [8]. However, the inclusion of PG can reduce the required water quantity for the setting reaction. This, in turn, minimizes the development of capillary pores within the cement structure and enhances the ultimate CS [9]. On the other hand, the presence of sufficient water is necessary to facilitate the hydration reaction of the cement. In cases of water inadequacy, the setting reaction may be interrupted, potentially compromising the CS. Collectively, increased CS of MTA following addition of PG remains to be a matter of debate and determining the proper PG ratio to incorporate in MTA composition remains to be elucidated [10, 11].

Calcium chloride (CaCl₂) is an inorganic compound that can be created by neutralizing hydrochloric acid with calcium hydroxide. It has been demonstrated that addition of this material to Portland cement can accelerate ST by facilitating the hydration reaction [12]. Conversly, it can also reduce the amount of water required for clinical applications. By the same token, inclusion of CaCl₂ can enhance CS of the cement via reducing its porosity [9]. This substance has been incorporated into MTA in varying proportions, as documented in the literature [9, 13, 14]. Nevertheless, consensus on the optimal percentage for the inclusion of CaCl2 remains a matter of debate.

Na₂HPO₄ is an inorganic compound with a pH of 9.5. This liquid can accelerate the ST of MTA by phosphate effect in cements with α-tricalcium phosphate as the powder [15]. Although several solutions and powders can provide phosphate, this formula has been suggested because of providing a more physiologic pH compared to other materials [15]. It has also been indicated that alkalinity of the environment can enhance the CS of MTA [16]. It should be noticed that any alteraltion in the setting process of CSCs can influence hydration products, which, in turn, significantly influence their biological effects.

Various studies have sporadically evaluated the effect of these substances on the mentioned properties of MTA, indicating contradictory results. Even in cases where the results of the studies were consistent, it was not possible to decide on a more appropriate concentration of the additive because heterogenous percentages of the material were examined in studies. In addition, due to a large number of studies on variable MTA properties, it was not possible to reach consensus about the effect of an additive on the most critical properties of MTA. Nevertheless, the incorporation of any substance into MTA may enhance certain properties while compromising others. To the best of the authors' knowledge, there is no comprehensive information about the effect of these additives on the critical properties of MTA. Therefore, the purpose of this systematic review was to investigate the effect of substances added to MTA on its physical, chemical, and cytotoxic properties. In case there was a consensus on the effectiveness of a certain additive on the cement in the literature, attempts have been made to provide a comprehensively reviewed information.

The present systematic review adheres to the guidelines outlined in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement [17]. Additionally, it has been registered in the International Prospective Register for Systematic Reviews (PROSPERO) under the registration number CRD42021253707.

The result of this meta-analysis revealed that the addition of PG increased the ST of MTA. This increase may result from chemical interaction between the PG and hydrating silicates and aluminate, which yields an amorphous materials in the cement matrix [40]. CS is an indicator of setting reaction. Since moisture is required for the setting reaction of MTA, addition of PG performs a fluidizing action on MTA-water mixture. Thus, a lower amount of water is required to reach a clinically acceptable consistency of MTA. Additionally, PG is a hygroscopic (water-absorbing) compound which can be added to the MTA powder. A reduction in water/powder ratio and water available for powder may decrease the CS of the cement [10, 40]. The results of the included studies were contradictory [10, 39], which can be due to the different experimental settings in the studies. Use of paraffin in the mold-sample interface probably to facilitate separation of the sample from the mold in one study [10], can be the culprit for lower CS after the addition of PG. Another difference between the two studies is the use of different liquids to keep the samples moist during the setting process (distilled water and phosphate-buffered saline). The measured CS of calcium silicate cements is directly influenced by the methodology of evaluation. In various studies, CS is measured after storage of samples in 100% humidity or complete immersion in an aqueous environment. The results of the study by Ha et al. [41] indicated that immersion of the sample into an aqueous environment results in higher CS values compared with those stored in high humidity. Since none of the included studies in this systematic review used water-immersed samples, lower CS values were reported compared with other similar investigations [41, 42].

Hydration products of MTA consist of calcium silicate hydrate (CSH) gel and calcium hydroxide. Ca²⁺ is produced in high proportions from calcium hydroxide and by the decomposition of CSH, which leads to an alkaline pH [43]. The addition of PG results in slower ST, and as a result, Ca²⁺ formation can be sustained for a more extended period which is in accordance with the studies included in this review [6, 8, 11]. If a high PG to water ratio is added to MTA, the water required for completing hydration reaction will be insufficient thereby minimizing formation of hydration products [11]. The Food and Drug Administration (FDA) has classified PG as an additive that is "generally recognized as safe" [44]. Two animal studies revealed that the addition of PG does not increase tissue inflammation more than the amount caused by MTA alone [6, 45].

Disodium hydrogen phosphate might increase the CS of MTA because of its pH. However, this was not in accordance with two similar original investigations [24, 32]. This solution can accelerate hydroxyapatite formation by providing phosphate [46]. Hydroxyapatite can promote cell adhesion and differentiation. Lotfi et al. evaluated inflammatory reactions following subcutaneous implantation of WMTA mixed with disodium hydrogen phosphate in an animal model [47]. The results revealed that addition of this substance reduced inflammatory reactions, but the difference between original and modified WMTA preparations was not significant. This finding was in accordance with the result of the current meta-analysis indicating that addition of 2.5% wt. Na₂HPO₄ to MTA does not adversely affect the biocompatibility of the material and might be able to promote further osteo/dentinogenic differentiation.

The result of this meta-analysis showed that addition of both 5% and 10% CaCl₂ to white MTA resulted in a reduction in both initial and final ST. Acceleration in setting corresponds with more formation of hydration products from the initial stages of setting reaction. This results in a decreased total porosity of admixture, leading to an increase of CS [12]. However, this was not in accordance with the studies included in this systematic review. This controversy may be due to the difference in the percentage of CaCl₂ added to MTA.

Addition of CaCl₂ can increase Ca²⁺ ion release and pH mainly because it provides additional Ca²⁺ ions for the cement [36]. However, the result of this meta-analysis indicates that this additive does not significantly alter the pH and Ca²⁺ release of MTA. This might be due to the different laboratory settings in the studies included. Unfortunately, there is no standard specification regarding the dimentional size of the specimens used to evaluate Ca²⁺ ion release and pH. Ca²⁺ ions may be responsible for biological effects of MTA [48]. Therefore, because CaCl₂ promotes Ca²⁺ ion release of the cement, it is expected that this admixture promotes cell viability and osteo/dentinogenic differentiation. McNamara et al. evaluated biocompatibility of 5% CaCl₂ added to MTA in an animal model [49]. The authors stated that at the 8th week, the inflammatory reaction of the CaCl₂ group was not statistically different from the control. The results of the present meta-analysis indicate that cell viability is not significantly affected by the addition of 5% CaCl₂ to MTA. However, adding 10% CaCl₂ reduces cell viability. It is possible that the increased quantity of chloride ions may adversely impact the biocompatibility of the cement [50]. Thus, lower percentages of CaCl₂ are recommended regarding biocompatibility of the cement. All studies included in this systematic review and meta-analysis evaluated cell biocompatibility via tetrazolium salts (MTT and XTT). These evaluations quantitatively assess mitochondrial activity, and the outcomes serve as a benchmark for cellular viability subsequent to exposure to test materials [51]. As stated previously, Ca²⁺ ions released from MTA can lead to odonto/osteogenic differentiation of stem cells, thereby resulting in the production of mineralized materials. In this regard, additional metabolic energy is supplied by enhanced mitochondrial activity as a prerequisite, which, on the other hand, can be misinterpreted as increased cellular proliferation when the cell viability is assessed using MTT or MTS assays. Therefore, it is recommended that viability assays other than use of tetrazolium salts be used to evaluate the cytotoxicity of substances that increase mitochondrial activity. It is important to notice that the findings of this study are applicable solely to the specific brands examined, and combining different additives may yield disparate results due to potential interactions among various materials.

Addition of 20% PG, 2.5% wt. Na₂HPO₄ and 5% CaCl2 to MTA are recommended to improve the admixture's physical, chemical, and cytotoxic properties.