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Acid-etching protocol for bioceramic cements: Evaluation of bond strength, compression and morphology

Paula Cosenza DDS, MSc1 | Ana Grasiela Limoeiro DDS, MSc, PhD2   |

Américo Bortolazzo Correr DDS, MSc, PhD7 | Alana Pinto Carôso Souza DDS, MSc7   |


1Department of Endodontics, São Leopoldo Mandic School, São Leopoldo Mandic Research Center, Campinas, São Paulo, Brazil

2Department of Dentistry, Endodontics and Dental Materials, Bauru Dental School, University of Sao Paulo, Bauru, Brazil

3Department of Endodontics and Dental Research, Iguaçu University, Nova Iguaçu, Brazil

4Department of Oral Health Sciences, BIOMAT – Biomaterials Research Group & UZ Leuven (University Hospitals Leuven), Dentistry, KU Leuven (University of Leuven), Leuven, Belgium

5Laboratory of Orofacial Pathologies, Imaging and Biotherapies, School of Dentistry, Laboratoire d'Excellence INFLAMEX, Université Paris Cité, URP 2496, Montrouge, France

6Department of Endodontics, Piracicaba Dental School, Campinas State University, Piracicaba, São Paulo, Brazil

7Department of Restorative Dentistry, Piracicaba Dental School, Campinas State University, Piracicaba, São Paulo, Brazil


Correspondence

Ana Grasiela Limoeiro, Department of Dentistry, Endodontics and Dental Materials, Bauru Dental School, University of São Paulo, Bauru, Brazil. Email: grasielalimoeiro@gmail.com


Abstract

To establish an acid-etching protocol for Biodentine and Cimmo DTA, evaluating compressive strength, bond strength, surface morphology in scanning electron mi- croscope and failure modes after different etching times. Two test specimens were prepared for each cement and divided into four groups (n = 12) according to the acid- etching time (0, 5, 10 and 15 s). Compressive strength was tested using a univer- sal testing machine, while bond strength was evaluated after bonding with Filtek Bulk Flow resin using Universal ESPE Single Bond adhesive. Failures were classi- fied as surface-adhesive, cement-cohesive, resin-cohesive and mixed. Biodentine showed significantly higher compressive strength than Cimmo DTA (p < 0.001), re- gardless of acid etch time (p < 0.001). Different acid-etching strategies are required for Biodentine and Cimmo DTA, with Biodentine requiring selective etching and Cimmo DTA requiring a full 15-s etch to optimise bond strength properties.


KEYWORDS

dental cements, root canal treatment, silicate cement

Aust Endod J. 2024;00:1–9. wileyonlinelibrary.com/journal/aej

© 2024 Australian Society of Endodontology Inc. | 1


INTRODUCTION


Bioceramic cements are widely used in endodontics to re- pair and fill canals, especially in complex cases such as perforations, internal root resorption or pulp capping/pul- potomy procedures [1]. Their biological properties include sealing capacity, bioactivity and induction of hard tissue, antibacterial activity, biocompatibility, fixation capacity in humid environments and low solubility [2, 3]. These cements, also referred to as hydraulic cements, calcium silicate-based cements, MTA and other names found in the literature [4], react in the presence of physiological fluids and form hydroxyapatite mainly on their surface, which induces the differentiation of dental pulp cells [2].

BiodentineTM (BD, Septodont, Saint-Maur-des-Fossés, France), one of these cements, is used as a dentin substitute in various endodontic applications, such as deep coronal restorations, deep cervical and root lesions, as well as pulp capping, pulpotomy, repair of root perforations, furcation perforations and resorptions [4]. BD is produced in the form of a powder of high-purity tricalcium silicate, which guar- antees that it does not contain aluminium inclusions and traces of metals present in Portland cement-based cements. In addition to BD, Cimmo-DTA cement (CDTA, Cimmo, Pouso Alegre, Brazil), which is also recom- mended as a dentin substitute for direct and indirect pulp

protection and pulpotomy, was also investigated [5].

This study is justified by the fact that it evaluates the be- haviour of the tested bioceramic cements when used as a protective shield and base for restorations, identifying the most appropriate protocol for this new reality of these ma- terials. In addition, to guide clinicians in the effective use of bioceramics to take advantage of their biological properties without compromising the final restorative technique.

Therefore, the aim of this study was to establish an acid- etching protocol with 37% phosphoric acid suitable for the surfaces of two bioceramic cements: Biodentine and Cimmo DTA, investigating the compressive and bond strength as well as the surface morphology and type of failure after dif- ferent etching times. The null hypotheses tested are that (i) the different etching times have no effect on the compres- sive strength of the bioceramics; (ii) the bond strength of the bioceramic cements is not altered by different etching times; (iii) the etching times do not cause changes in the mi- cromorphology of the bioceramic cements; (iv) the mode of failure and bond strength are not affected by etching.



MATERIALS AND METHODS


As no biological samples were used in this study, it was exempted from submission to the Research Ethics Committee (Protocol No. 2023-0085).


The manuscript of this laboratory study was written according to the Preferred Reporting Items for Laboratory studies in Endodontology (PRILE) 2021 guidelines [6] (Figure 1).

The sample size calculation was based on finding a large effect size based on the data published by [7]. To achieve a power of 90% and a significance level of 5 with a large effect size of 0.40 [8], the program G*Power, ver- sion 3.1.9.4, indicated according to the factorial analysis of variance model that 12 samples per group are required for each of the power tests.

The composition of the bioceramic cements investi- gated in this study is described in Table 1.



Preparation of the specimens for the compressive strength test (sample 1)


Ninety-six cylindrical plastic moulds with a diameter of 6 mm and a height of 6 mm were prepared from poly- propylene cylinders (measured with a precision ver- nier calliper) and cut with a 15C carbon steel scalpel tip (Swann-Morton Ltd., UK), which served as the mould for the bioceramics. This was inserted and homogeneously compacted with an Almore – Suprafil 1/2 resin spatula (Quinelato Industria e Comércio Ltda, Rio Claro, Brazil). During the first 12 min of curing, the bodies were pressed onto their surface with a weight of 500 mg to ensure a flat surface. The samples were covered with a damp absorbent cotton cloth, placed in a closed Eppendorf and stored in an ECB 1.3 digital oven (Odontobras Ind, e Comércio Odont. Ltda, Ribeirão Preto, Brazil) with 99%–100% humidity at 37°C for 72 h. After this time, the moulds were removed, and the samples were weighed on a precision balance to ensure that all groups had the same weight and standardi- sation was guaranteed (Figure 2).



Preparation of the samples for the universal adhesive bond strength test (sample 2)


Ninety-six specimens with a diameter of 6 mm and a height of 3 mm were printed in holes in resin blocks specially manufactured for this project by Acrilaser – Acrílicos (São José do Rio Preto, Brazil) (Figure 3).

The cements were prepared according to the manufac- turer's recommendations and immediately inserted into the holes in the blocks. During the first 12 min of setting, the samples were loaded with a weight of 500 mg to en- sure the most even and homogeneous surface possible. The specimens were stored in a closed plastic container, where they remained at a humidity of 99%–100% at 37°C in an ECB 1.3 digital oven (Odontobras, Tabatinga, Brazil).



FIGURE 1 PRILE flowchart.


All specimens with the appropriate characteristics (di- ameter and width), without internal defects and homo- geneous, were included in the study. All specimens with manufacturing defects were excluded.


Acid conditioning


Specimens 1 and 2 were divided into eight groups each (n = 12) and subjected to four acid etchings (0, 5, 10 and


TABLE 1 Composition of Biodentine and Cimmo-DTA cements.


Bioceramic cement BD CDTA

Powder Tricalcium silicate

Dicalcium silicate Calcium oxide Calcium carbonate Zirconium oxide




Liquid Calcium chloride

Dicalcium silicate Calcium carbonate Pozzolone Calcium Aluminium Potassium Magnesium Barium sulphate

Distilled water

Polycarboxylate

Abbreviations: BD, biodentine; CDTA, Cimmo DTA.


FIGURE 2 Specimen made for the compressive strength test.




FIGURE 3 Specimen for the universal adhesive bond strength test.


15 s) (Table 2), using 0.025 mL of 37% Condac phosphoric acid (FGM Indústria e Comércio de Medicamentos Ltda, Sabaudia, Brazil). The acid was applied to one of the sur- faces using a 0.5 mL insulin syringe 6 mm × 0.25 mm ul- trafine 50ui Sr. – 200 u (SR—Saldanha Rodrigues Produtos Hospitaleres LTDA, Manaus, Brazil).

After the conditioning time, the samples were washed for 15 s with 3 mL of saline (volume controlled by a per- istaltic pump with a flow rate of 6 mL/min) (LAP-101-3

Tecnopon, Piracicaba, Brazil) and dried for 15 s using a tri- ple syringe at 30 mm from the sample.



Evaluation of the scanning electron microscope (SEM)


Another unit of sample 1 (8 units) from each experimen- tal group was prepared for SEM evaluation (Figure 4) to check the micromorphology of the surface (formation of pores, microchannels, dissolved crystals or obvious cracks).



Testing the compressive strength


The compressive strength was determined using an EMIC DL 2000 universal testing machine (INSTRON BRASIL Equipamentos Científicos Ltda., São José dos Pinhais, Brazil). The maximum force at the time of frac- ture was recorded using a 2000 kgf load cell. To apply this force, a blunt tip with a cylindrical active end with a diameter of 3.6 mm and a length of 16 mm was placed on


TABLE 2 Distribution of groups according to acid-etching time.



Samples 1




the surface of the specimen in the direction of its longi- tudinal axis perpendicular to the ground. The compres- sive load was applied at a constant rate of 1.0 mm/min until the specimen fractured. The breaking force was given in Newtons (N).



Bond strength test


A drop of Single Bond Universal ESPE Adhesive (3 M do Brasil Ltda, Sumaré, Brazil) was placed in a dab pot and applied in a thin layer to the surface of specimen 2 using a standard Micro Brush (KG – SORENSEN, Cotia, Brazil), gently air-dried for 10 s and photoactivated with the VALO Cordless light-curing device (Ultradent do Brasil Produtos Odontológicos Ltda, Indaiatuba, Brazil) at a light intensity of 1000 mW/cm2 in continuous mode for 10 s.

Subsequently, a polypropylene plastic cylinder with a diameter of 6 mm and a height of 3 mm was positioned

37% phosphoric acid

BD CDTA

over the bioceramic cement and Filtek Bulk Flow Resin


No conditioning BD1 NC CDTA1 NC

5 s BD1 5 s CDTA1 5 s


10 s

15 s

BD1 10 s

BD1 15 s

CDTA1 10 s

CDTA1 15 s



Samples 2



37% phosphoric acid

BD

CDTA


No conditioning

BD2 SCA

CDTA2 SCA


5 s

BD2 5 seg

CDTA2 5 seg


10 s

BD2

10 seg

CDTA2 10 seg


15 s

BD2

15 seg

CDTA2 15 seg


Abbreviations: BD, biodentine; CDTA, Cimmo DTA; NC, no conditioning.

(3M do Brasil) was applied in contact with the cement cov- ered with Universal Adhesive. The procedure was carried out in two increments, the first being 1 mm high and the second 2 mm high. Polymerisation was carried out for 20 s in the same way as for the adhesive layers. At the end of polymerisation, the second mould around the polymer- ised resin was removed and we had a resin body bonded to sample 2.

Sample 2 was stored like sample 1 in an incubator with a humidity of 99%–100% and a temperature of 37°C for 24 h. The bond strength was tested using the same univer- sal machine at a speed of 1 mm/min and the values were recorded in Newtons and converted to MPa. The loose sur- faces were then evaluated under an optical microscope at 20× magnification to classify the types of failure.


FIGURE 4 SEM images of the different acid etching times tested.

(a–d) Biodentine; (e–h) CIMMO DTA; a and e—no acid etching; b and f—5 s of acid etching; c and g—10 s of acid etching; d and h—15 s of acid etching.


Statistical evaluation

After verifying compliance with the normal distribu- tion and homoscedasticity, the compressive and bond strength data were subjected to a generalised linear

TABLE 3 Means (standard deviations) of compressive strength (Newton) and bond strength (MPa) of bioceramic materials, according to acid-etching protocol.



Compressive strength

Overall

model to investigate the effects of the bioceramic ma-

Acid

BD CDTA

average














than that of CDTA (p < 0.001), but neither was statistically significantly affected by acid etching (p = 0.923) (Table 3). There was a significant difference in bond strength between the type of cement and the time of acid etching (p < 0.001). The Tukey test revealed that BD resulted in significantly higher bond strength than CDTA, regardless of acid application. For BD, the highest values for bond strength were found after 5 or 10 s of acid etching. Without the application of acid, BD showed significantly higher bond strength than when the acid was applied for 15 s. This conditioning time resulted in significantly higher bond strength values for CDTA. The groups without etching or with etching for 5 or 10 s showed significantly lower values, but no significant difference between them

(Table 3).

Regarding the mode of failure, the cements tested without acid (p = 0.373) or with an etching time of 5 s (p = 0.524) and 15 s (p = 0.700) showed no significant dif- ference. However, there was a difference between the two materials at an application time of 10 s (p = 0.020). At this point, cohesive failures predominated in the cement of BD, while CDTA had a higher proportion of mixed fail- ures. No significant difference in failure modes was ob- served for BD (p = 0.139) or CDTA (p = 0.332) when acid etching was not performed or when it was performed for 5, 10 or 15 s.

Biodentine showed uniform microstructures similar to crystalline formation even without acid etching, with greater clarity on surfaces etched for 5 and 10 s. However, conditioning for 15 s resulted in the formation of voids, indicating a possible detrimental effect on the surface of the material. In contrast, the unconditioned surfaces in CDTA do not exhibit uniform microretentions, which are clearly different from a crystalline formation. However, with 5- and 10-s conditioning, microdeformations begin



Acid


BD


CDTA

Overall

average

No conditioning

4.56

3.89 (0.90) Bb

¾


(1.35)

Ab



5 s

5.43

4.09 (0.87) Bb

¾


(1.01)

Aa



10 s

5.25

4.11 (0.80) Bb

¾


(0.77)

Aa



15 s

4.21

5.95 (0.86) Ba

¾


(0.68)

Ac



Note: For the compression data, overall averages followed by different capital letters indicate a significant difference between cements. For the compression data, overall averages followed by equal lowercase letters indicate no significant difference between samples submitted or not to the acid application times. For the shear data, means followed by

different capital letters indicate a significant difference between cements (comparisons within each row). For the shear data, means followed by different lowercase letters indicate a significant difference between acid application times (comparisons within each column).



to manifest, while 15 s conditioning leads to crystalline formations with no recognisable voids.



DISCUSSION


The compressive strength of bioceramic cements is fun- damental to their effectiveness and is influenced by the hydration process during setting [9, 10]. In addition to compressive strength, it is crucial to consider the inter- action between the bioceramic and the materials used for the aesthetic restoration, such as universal adhesives,whose performance is not affected by acid etching. The aim of this study was to establish a suitable acid-etching protocol for BD and CDTA bioceramic cements, to eval- uate their compressive and bond strength and to analyse the surface morphology and failure modes after different acid-etching periods. The null hypotheses tested were:

(i) different acid-etching times do not affect the com- pressive strength of bioceramics; (ii) the bond strength of bioceramic cements is not affected by different acid- etching times; (iii) acid-etching times do not cause changes in the micromorphology of bioceramic cements;

(iv) the type of failure and bond strength are not affected by acid etching.

In this study, Single Bond Universal ESPE Adhesive (3M do Brasil Ltda, Sumaré Brasil) was used, whose for- mulation consists of various components, including bi- sphenol A diglycidyl ether dimethacrylate (BIS-GMA), 2-hydroxyethyl methacrylate, silicone-treated silicon dioxide, ethyl alcohol, decamethylene dimethacrylate, water, 1,10-decanediol phosphate methacrylate, copo- lymer of acrylic and itaconic acid, camphorquinone, N,N-dimethylbenzocaine, 2-dimethylamonoethyl meth- acrylate, methyl ethyl ketone. These adhesives are self- etching in one step and their adhesion and clinical performance is independent of acid etching [11].

Some studies consider that better bonding results are obtained if the restorative treatment is performed 7 days after the application of the bioceramic cement [12, 13]. Other authors have evaluated the effects of surface treat- ments of tricalcium silicate-based bioactive restorative materials on bond strength to composite resin and have concluded that BD appears to be more suitable for use in acidic environments [14] but that the microhardness of BD decreased when exposed to 35% phosphoric acid for 1 min [2].

Since there is no consensus on which adhesive system is more advantageous when bonding bioceramics to res- ins, in this study we opted for a system in which the same adhesive was used in all groups to eliminate the effects of chemical composition and focus on one variable, namely acid etching. The choice of universal adhesive was based on a previous study [15] in which a higher bond strength of BD was observed with this type of adhesive. This can be attributed to the porous nature of BD and the pres- ence of the functional monomer 10-MDP in the universal adhesive, which enables chemical bonding to the oxides present in the biomaterial [16]. A study [13] reported that there was no significant difference in the adhesion

test for BD with values of 4.44 ± 2.49 and 3.09 ± 2.23 MPa after 12 min and 7 days, respectively [16]. In contrast,

another study [17] observed the highest adhesion value (32.47 ± 8.18 MPa) for the 14-day group and the lowest (4.08 ± 0.81 MPa) for the 12-day group.

In the present study, Condac 37% phosphoric acid was chosen as the etchant, which is characterised by a low- viscosity aqueous base with thixotropic properties. It can be used on both dentin and enamel for a period of 15 s, which is facilitated by the blue dye that allows control during application. Filtek Bulk Fill Flowable resin has been used in all groups to eliminate the effects of chem- ical composition and focus on acid etching as the main variable. This material was selected for its composition combining filler particles and monomers to maximise mechanical strength, wear resistance, radiopacity and re- duced polymerisation shrinkage.

The first null hypothesis was confirmed as there was no significant interaction between the tested bioceramics and acid etch time in terms of compressive strength. BD had a higher compressive strength than CDTA, but acid etching did not change this situation positively or nega- tively for either of the two bioceramics tested. Both ma- terials maintained their strength at all acid-etching times, possibly due to the presence of calcium carbonate [7] and calcium chloride [18] which shorten the setting time and may make them less sensitive to acid damage. In addition, the polycarboxylate contained in Biodentine may have contributed to this effect.

On the other hand, the second null hypothesis was re- jected, as BD performed better at 5 and 10 s, while con- ditioning for 15 s led to the worst results, possibly due to the presence of unique structures on the surface of bioc- eramics after curing [19, 20]. The different behaviour of BD and CDTA in a 15-s acid etch may be related to the differences in their compositions (Table 2), with BD con- taining polycarboxylate, which may affect its solubility in contact with acids. The low pH of phosphoric acid may affect the chemical bonding process by disrupting the hydration of tricalcium silicates in BD and negatively af- fecting its microstructure [21]. In this scenario, it could be hypothesized that the polycarboxylate polymer would form “deposits” that would be removed upon contact with 37% phosphoric acid for 15 s. On the other hand, CDTA, which is biomimetic with respect to its starting material, shows a different reaction.

The duration of acid etching can cause micromor- phological changes to the surfaces of materials [20], with CDTA showing greater resistance to adhesion at longer etching times (15 s) in contrast to BD. Increasing the acid-etching time resulted in poorer micromorphol- ogy in BD, whereas in CDTA the surface was more ho- mogeneous, which could explain the better adhesion resistance. The development of these microporosities, as in enamel conditioned with 37% phosphoric acid for 15 s, would explain the good result of CDTA in the bond strength test. Therefore, the third null hypothesis was rejected, indicating a significant change in surface micromorphology as a function of acid-etching time. These data confirm the results of a study in which the effect of 37% orthophosphoric acid on BD was investi- gated using SEM [2]. It was observed that after 20 s of acid attack, structural and chemical changes occurred in Biodentine that may affect its bond strength with a resin composite material [2].

The fourth null hypothesis was accepted as no signif- icant correlation was found between the failure modes and adhesion strength between the groups of the same biomaterial and between the two cements. These results agree with those of a study [22], that investigated different surface treatments of bioceramic cements. These authors found that the use of dentin adhesives did not result in an effective bond line. Even with the additional use of phos- phoric acid etching, there was no improvement in bond strength. The authors also pointed out that longer etching times for BD, exceeding 30 s, may have a negative impact on bond strength and properties when self-etching tech- niques are used.

However, the study had limitations, such as the lack of inclusion of all variables of a clinical situation, such as polymerisation stress and pH changes, thermal and load- ing cycles and enzymatic challenges that may affect the adhesive-cement interface. Therefore, further long-term studies are required to confirm the results.

We can conclude that different acid-etching strategies are required for Biodentine and Cimmo DTA, with selec- tive etch for the former and full 15-s etch for the latter, to optimise their bond strength properties.


AUTHOR CONTRIBUTIONS

Conceptualization: Paula Consenza and Aline Cristine Gomes Matta; Data curation: Marcos Frozoni and Alana Pinto Carôso Souza; Formal analysis: Marcos Frozoni and Américo Bortolazzo Correr; Investigation: Paula Consenza and Adriana de Jesus Soares; Methodology: Ana Grasiela Limoeiro; Wayne Martins Nascimento; Marilia Fagury Videira Marceliano-Alves and Américo Bortolazzo Correr; Project administration: Ana Grasiela Limoeiro and Wayne Martins Nascimento; Supervision: Ana Grasiela Limoeiro and Wayne Martins Nascimento; Validation: all authors; Visualization: all authors; Writing – original draft: all authors; Writing – review and editing: Ana Grasiela Limoeiro and Paula Consenza


CONFLICT OF INTEREST STATEMENT

The authors deny any conflicts of interest related to this study.


ORCID

Wayne Martins Nascimento  https://orcid. org/0000-0003-4201-4710

Marilia Fagury Videira Marceliano-Alves https://orcid. org/0000-0002-2917-5934

Adriana de Jesus Soares https://orcid. org/0000-0002-8078-1606

Américo Bortolazzo Correr https://orcid. org/0000-0002-3306-7055

Alana Pinto Carôso Souza https://orcid. org/0000-0002-2743-3012

Aline Cristine Gomes Matta https://orcid. org/0000-0003-2789-5185

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