Temporal effects of restorative pretreatments on Biodentine: an in-vitro study

Authors: Akshaya Narendrakumar, Hrudi Sundar Sahoo, Kurinji Amalavathy Ratnakaran, Adisree Ravichandran

Abstract

Background

Biodentine is frequently exposed to various surface pretreatments, such as dentin conditioner (DC), glass ionomer liquid (GICL), acid etchant, and adhesives, during pulp capping procedures. However, the impact of these agents on Biodentine’s surface microstructure and chemical composition remains underexplored.

Objective

This study qualitatively analyzed the effects of DC, type II GICL, acid etchant (AE), Clearfil SE (CFS, a two-step self-etch adhesive), and Single Bond Universal (SBU, a universal adhesive) on the surface microstructure and chemical composition of Biodentine (BD) at 5 min, 12 min, 24 h, and 7 days after manipulation.

Materials and methods

BD samples were subjected to the pretreatments and qualitatively analyzed using scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDX), micro-Raman spectroscopy, and X-ray diffraction (XRD).

Results

SEM revealed distinct surface morphologies depending on the pretreatment and time interval, ranging from homogenous and grainy to smooth and polymer-coated surfaces. EDX showed significant variations in calcium-to-silicon ratios over time. Notably, AE caused pronounced surface disruption up to 24 h, while CFS and SBU resulted in minimal changes. DC, GICL, and DC+GICL did not adversely affect BD’s surface microstructure at any time point. XRD and micro-Raman analyses indicated no change in the chemical composition of BD across all groups and time intervals.

Conclusion

Surface pretreatments influence BD differently depending on timing. AE compromised surface integrity until 24 h, whereas self-etch and universal adhesives produced only superficial alterations. Delaying aggressive pretreatments until 7 days minimizes disruption.

Introduction

Biodentine (BD) represents a significant advancement in calcium silicate-based biomaterials, serving as a bioactive tricalcium silicate cement specifically engineered as a dentin substitute for diverse endodontic applications [1, 2]. This innovative material demonstrates exceptional biocompatibility and bioactivity, effectively stimulating pulp healing through the induction of tertiary dentin formation while maintaining pulp vitality in direct and indirect pulp capping procedures [3, 4]. The clinical significance of BD extends beyond its bioactive properties to encompass its role in successful restoration outcomes, where the quality and longevity of overlying restorative materials are critically dependent on proper surface treatment protocols and optimal timing of restoration placement [5–8].

Contemporary research has extensively documented BD’s unique composition consisting primarily of tricalcium silicate (80.1%), zirconium oxide as a radiopacifier, and calcium chloride as a setting accelerator, resulting in an initial setting time of approximately 12 min [7–9]. The material’s hydration mechanism produces calcium silicate (CS) hydrate gel and calcium hydroxide (CH), establishing an alkaline environment (pH 12) that promotes antimicrobial activity and facilitates biomineralization processes [10, 11]. However, despite its relatively rapid initial setting, BD requires an extended maturation period of 14–28 days to achieve optimal mechanical properties and complete crystallization of the CS hydrate matrix [12, 13]. Current clinical protocols regarding the timing of surface pretreatments and definitive restoration placement demonstrate considerable variability, with some practitioners advocating immediate restoration after initial setting, while others recommend delayed approaches ranging from 24 h to several weeks [5]. The role of surface pretreatments, including acid etching, sandblasting, and chemical conditioning, has been established as crucial for enhancing micromechanical retention and improving bond strength to overlying composite restorations [6, 14].

Despite extensive clinical validation of BD’s efficacy in vital pulp therapy, significant knowledge gaps persist regarding the temporal effects of restorative pretreatments on the material’s surface microstructure and chemical composition [15, 16]. Current literature predominantly focuses on immediate or short-term bond strength assessments, with limited investigation into the qualitative changes occurring at the BD surface following various pretreatment protocols over extended time periods [17, 18]. The dynamic nature of CS cement maturation suggests that surface characteristics may undergo continuous modification, potentially affecting the efficacy of pretreatment procedures applied at different maturation stages [19–21]. The absence of systematic investigation into temporal surface changes represents a critical limitation in understanding how pretreatment timing influences the long-term stability and performance of BD-composite interfaces [12, 20]. Thus, clarifying time-dependent surface microstructural and chemical alterations in BD subsequent to standard restorative pretreatment protocols is clinically significant for guiding decisions regarding immediate versus deferred definitive restoration placement.

The aim of this study was to qualitatively analyze the effect of pretreatments for glass ionomer restoration (GIC) and composite restoration on the surface microstructure and chemical composition of BD at various time points after manipulation – 5 min, 12 min, 24 h and 7 days. The pretreatments for the GIC restoration involved in this study were the application of dentin conditioner (DC), type II glass ionomer liquid (GICL), and combination (DC+GICL). The pretreatments for composite restoration were the application of phosphoric acid etching (AE), Clearfil SE (CFS), and Single Bond Universal (SBU) in self-etch mode. The study’s null hypothesis was that the surface microstructure and chemical composition of the BD would not be affected by the different pretreatments at various time points after manipulation.

Materials and methodology

The study was approved by the Institutional Review Board and Institutional Biosafety and Ethical Committee (IBEC), SIST. IRB 167/IRB-IBSEC/SIST. Eighty-four standardized BD discs (10 mm × 2 mm) were prepared by mixing BD powder and liquid (Septodont, St. Maur-des-Fossés, France) in an amalgamator according to the manufacturer’s instructions [22]. The material was overfilled into stainless-steel molds and allowed to set under controlled conditions (37°C, 100% humidity). The BD discs were subjected to assigned pretreatments at 5 min, 12 min, 24 h, and 7 days after manipulation (n = 4). The surface pretreatment agents (Table 1) included DC containing 10% polyacrylic acid (GC Corporation, Tokyo, Japan), Type II glass ionomer cement liquid (GC Fuji II, GC Corporation, Tokyo, Japan), and a combination of DC followed by GICL. For composite-related pretreatments, 37% orthophosphoric acid etchant (D-Tech Etching Gel, D-Tech Technologies, India), Clearfil™ SE Bond (Kuraray Noritake Dental Inc., Tokyo, Japan), and Single Bond Universal (3M ESPE, St. Paul, MN, USA) were used. The BD samples were stored in an incubator at 37°C and 100% humidity until being subjected to pretreatment. SEM-EDX (Zeiss MERLIN Field Emission SEM, Carl Zeiss NTS GmbH, Oberkochen, Germany), X-ray diffraction (XRD) (Rigaku, Tokyo, Japan), and Micro-Raman spectroscopic (Renishaw plc, Wotton-under-Edge, UK) analyses were performed according to previously published research studies [17]. Figure 1 provides a comprehensive flow diagram outlining the sequential methodology adopted in this study. Table 1 presents an in-depth overview of the different restorative pretreatments evaluated in this study.

As this was a qualitative characterization study, no statistical analysis was applied. Surface changes were documented descriptively. Importantly, all analytical procedures (SEM-EDX, XRD, and Micro-Raman spectroscopy) were performed by independent, trained operators who provided their respective observation reports. These reports were collected by the authors and collated to generate the consolidated results presented in this manuscript.

Results

SEM-EDX analysis for surface characterization

SEM images obtained at 1,000x and 10,000x were used to analyze the surface morphology of the control and pretreated BD samples. The surface morphology as observed (Figures 2–4) under SEM is summarized in Table 2, and the calcium-to-silicon ratio obtained from EDX analysis (Figures 5 and 6) is listed in Table 3. The SEM and the EDX could not be performed immediately, at 5 min from the time of manipulation and pretreatment, due to technical limitations. Hence, the 5-min pretreatment samples were analyzed after 12 min. At 24 h, SEM images demonstrated intermediate morphological features between early (12 min) and late (7 days) observations. Control and DC groups showed more coalesced crystalline structures compared with the early phase, while AE continued to exhibit disrupted surfaces. CFS and SBU presented smoother coatings with voids, consistent with polymer coverage. As these patterns largely paralleled those at 12 min and 7 days, the full image set is included in supplementary data. EDX analysis was performed to evaluate the calcium-to-silicon (Ca/Si) ratio of BD surfaces following various pretreatments at different time points (Table 3). Overall, EDX analysis demonstrated that AE had the most detrimental effect on surface calcium content, while DC, GICL, and DC+GICL preserved the elemental composition of BD over time.

XRD analysis

The data were acquired in the form of graphs with peaks that represented the crystalline phases in the samples (Figure 7). All the samples matched with JCPDS-ICDD # 31-0301, which confirmed the tricalcium silicate presence, a triclinic phase with a lattice parameter of a = 14.013 Å, b = 14.210 Å, and c = 25.100 Å with a space group of p1(1) (11). The diffraction peaks observed at 29.51, 32.06, 32.32, 32.70, and 34.73 2θ can be, respectively, indexed as (401), (009), (044), (322), and (445) planes of BD. The peaks at 29.51 2θ represent calcium carbonate (CC), 32.06 2θ, 32.32 2θ, and 32.7 2θ represent CSs, and 34.73 2θ represents CH. All the control and pretreated BD samples showed peaks for CC, CS, and CH at all tested times. A broad peak at around 20° shows the presence of an amorphous phase. A summary of the analysis is provided in Table 4.

Micro-Raman spectroscopy analysis

A comprehensive interpretation of the molecular structure and composition of the sample observed in Micro-Raman Spectra (Figure 8) is depicted in Tables 5 and 6.

For brevity, only representative SEM micrographs, EDX spectra, XRD, and Raman patterns have been included in the main text. Additional datasets, including SEM images at 24 h, EDX spectra at 5 min and 24 h, and Raman spectra at 24 h, are available in the research data provided at the end of this manuscript.

Discussion

In clinical practice, definitive restorations are often placed immediately, sometimes before BD has set fully. To simulate this, pretreatments were applied at 5 min, 12 min, 24 h, and 7 days [5]. SEM images suggested varying degrees of disruption on the BD surface compared to the controls. At 5 and 12 min, all the surface pretreatments either inflicted etching, porosities, or erosion on the BD samples, but the effect was more pronounced with pretreatments for composite, especially with AE. All surface pretreatments for GIC restoration showed only mild surface disruption at all times tested. Bolhari et al. [21] suggested that these surface disruptions may have occurred due to calcium and zirconium depletion, and the same was confirmed from the results of EDX analysis in this study.

Phosphoric acid etching before 12 min caused erosion of the matrix, which reflected as a considerable reduction in the calcium-to-silicon ratio compared to the control in EDX. This reduction in the calcium-to-silicon ratio could drastically affect the formation of CS hydrate gel, which may adversely impact the surface hardness. Defects such as cracks, craters, and crumbled surfaces that appeared with phosphoric acid etching at 24 h seemed to be inconsistent with that of the intended effect. Our findings are in correlation with previous bond strength studies of BD to composite resin using ER adhesives [23–25]. However, Cengiz et al. reported a higher bond strength of BD to composite resin after immediate layering using ER adhesive and attributed the same to micro-mechanical bonding, but the present study showed conflicting evidence where surface pretreatment with phosphoric acid etching at 24 h or prior resulted in poor surface characteristics unsuited for micro-mechanical bonding [26]. At 7 days, phosphoric AE pretreatment produced microporosities uniformly spread across the BD surface. The surface damages with other pretreatments were minimal at 24 h and negligible at 7 days.

The EDX of the control samples was in line with previous studies [17, 21]. Additionally, it also revealed that control samples of BD showed the presence of CS hydrate gel with CC interspersed between them. The control samples demonstrated a high to low calcium-to-silicon ratio with time. The initial high could be attributed to the presence of unreacted CS hydrate gel. The needle-like crystal growth observed under SEM at 24 h, indicative of the initiation of the crystallization process, coincided with a low calcium-to-silicon ratio in EDX. The ongoing maturation process was demonstrated by a further reduction in the calcium-to-silicate ratio on the 7th day. This was possibly the result of CC formation over the calcium silicate hydrate (CSH) gel. Previous literature has shown that CC and CSH gel in BD are responsible for its hardness [27, 28]. This may be the reason that on the 7th day, the BD samples were least affected by any of the surface pretreatments. DC, GICL, and DC+GICL increased the calcium-to-silicon ratio, indicating a possible leaching of calcium ions from the surface of BD. In the present study, it was also observed that the calcium-to-silicon ratio was drastically reduced after pretreatment with CFS and SBU at all time points, which could be attributed to the interaction of calcium ions in the BD with 10-methacryloyloxydecyl dihydrogen phosphate (10-MDP) present in these adhesives [29].

Raman spectroscopy involves a noninvasive analytic approach for chemical identification and quantification [30]. Although specific sample preparation was not needed, the heat induced by the focused laser may damage the specimen’s surface up to a certain depth [31]. In this study, the power setting of the laser was reduced to 50 mV in order to avoid any thermal damage to the samples. At 24 h, these peaks were present but at a lower intensity, indicating progress in the setting reaction. The peak intensity of silicates and calcites was further reduced with the appearance of CH peaks on the 7th day. This was representative of the slow, sustained, and continuing phase of the setting reaction. This finding was in line with the FT-IR characterization done by Alotaibi et al. [19].

When the surface was pretreated with CFS and SBU adhesives, peaks appeared at 1,768 cm^-1^, indicating the presence of reaction byproducts characterized by ester bonds at all the tested times. This finding is in line with a previous study by Anastasiadis et al. who also demonstrated the formation of ester groups as a result of a chemical interaction between 10-MDP molecules and calcium in BD [32]. Interestingly, when the surface was pretreated with AE on the 7th day, no silicate peaks, very short and broad calcite peaks, and short and sharp CH peaks were observed. Camilleri et al. investigated the effect of acid etching on BD using FT-IR; their observations showed no change in the FT-IR peaks between the etched and unetched BD. Their finding was contradictory to the micro-Raman findings of the present study, but it is important to note that the authors failed to report the time of pretreatment in their study [17]. Although the SEM-EDX and Raman spectroscopic analyses showed considerable changes in the surface microstructure, the chemical composition within the sample remained stable. The XRD analysis of BD after various surface pretreatments revealed no change in the chemical composition. Hence, the null hypothesis was partially rejected. From the results of the present study, 37% orthophosphoric acid etching appears highly detrimental to the surface of BD until 7 days after mixing. Few studies have demonstrated a higher bond strength of BD to composites when delayed layering is done [12, 14, 21], and a few others in the literature also suggest poor bond strength between BD and composite resin, irrespective of the time of overlayering [23, 24, 26].

Correlating with the results of this study, in cases where an immediate overlay is required, GIC or composite resin restoration with mildly acidic self-etch adhesives may be the choice for a definitive restoration. The evidence on the quality of the bond between BD and composites is still sparse. Studies reported premature cohesive failure within BD when early overlayering with composite was performed, which suggests that BD may be a weak substrate for micromechanical bonding during the initial phase of setting and might not be able to resist the stress of polymerization shrinkage when early bonding is attempted [12, 24, 33]. Few limitations of this as the samples could not be sufficiently sputter coated due to the initial hydration in the 5-min sample, it posed difficulties in scanning the surface. Hence, the pretreated samples at 5 min could not be subjected to SEM-EDX analysis; rather, scanning was done only after 12 min. Then, the retention of certain pretreatment agents such as GICL, CFS, and SBU on the surface of the sample hindered the thorough analysis of their effect on the immediate underlying surface of BD. Further research is needed to evaluate the effect of various surface pretreatments on the physical and interfacial properties of BD.

Conclusion

Within the limitations of this study, it is concluded that BD showed surface microstructural changes after various surface pretreatments at all time points tested. None of the surface pretreatments tested adversely affected the surface microstructure or composition at the 7th day except for AE. AE was the only surface pretreatment that had an adverse effect on the surface microstructure and composition at all the time points tested before the 7th day. None of the surface pretreatments induced any change in the chemical composition during the setting and maturation of BD. The effect of these surface pretreatments must be further correlated with interfacial analysis and bond strength studies to make clinically relevant recommendations for the placement of definitive restorations.