Role of phosphate-buffered saline on push-out bond strength of MTA Flow™ and Biodentine™ after acid challenge: An in vitro study.

Context: The physical and chemical properties of root repair materials are adversely affected when placed in areas of inflammation with acidic pH. Aim: To evaluate the role of phosphate-buffered saline (PBS- pH 7.4) on push-out bond strength (POBS) of MTA flow and Biodentine (BD) after acid challenge with butyric acid buffered solution (BABS- pH 5.4). Subjects and
Methods: Eighty mid-root dentin slices (2 mm thick; 1.3 mm lumen diameter) were prepared and were divided into two groups (n = 40) based on the type of material used for filling lumen: Group 1-MTA Flow and Group 2-BD. Each group was again divided into four subgroups (n = 10) based on the duration of exposure to storage media: (a) 3 days in PBS, (b) 3 days in BABS, (c) 3 days in BABS followed by 30 days in PBS, and (d) 33 days in PBS. POBS was then measured using the universal strength testing machine. Statistical Analysis Used: Statistical analysis was performed with one-way analysis of variance and post hoc test using SPSS software version 23.0.
Results: Group 1b and 2b showed significantly lower bond strength values. No significant difference was observed between Group 1b and Group 1c (P > 0.05), whereas highly significant POBS values were observed between Group 2b and Group 2c (P = 0.000). Among all the tested groups, Group 2d showed the highest POBS values.
Conclusion: On storage in PBS after acid challenge, BD attained the highest POBS values while no significant difference was observed in MTA Flow. Copyright:

INTRODUCTION

Calcium silicate-based materials (CSBM) when applied as either retrograde filling in areas with active infection or as an orthograde apical plug in open apices with necrotic teeth, they are placed in an environment in which inflammation may be present and the surface of the unset material will be exposed to a low pH environment. The physicomechanical properties of CSBM were adversely affected when placed in areas of inflammation with acidic pH.[1] MTA exhibited increased solubility, decreased microhardness, and adhesion to root dentin on exposure to a pH of 5.[23] Biodentine (BD) exhibited higher surface hardness, compressive strength, and bond strength to the root dentin than White MTA when exposed to pH values of 7.4, 6.4, 5.4, and 4.4 due to its prominent biomineralizing ability.[14]

MTA Flow (Ultradent, South Jordan, UT, USA) was recently introduced as an alternative to conventional MTA with similar composition, smaller particle size (<10 μ), and shorter setting time of about 15 min.[5] It is supplied with an anti-washout water-soluble silicon-based gel which improved its compressive strength, reduced porosities, and is easily injected into the cavity.[6] The cytotoxic evaluation showed more fibroblasts with fewer inflammatory cells around MTA Flow. In contrast, BD showed a severe inflammatory response due to the presence of a modified polycarboxylate in its liquid.[5] MTA Flow showed apatite formation in simulated body fluids, indicating its bioactivity.[7] However, the effect of the acidic environment on the bond strength of MTA flow to dentin has not been studied.

Therefore, this in vitro study mainly aimed to evaluate and compare the push-out bond strength (POBS) of MTA Flow and BD to root dentin after exposure to acidic pH for the first 3 days and to investigate the role of phosphate-buffered saline (PBS) on these two materials after acid challenge.

SUBJECTS AND METHODS

The materials used in the study are shown in Table 1. With the power of study set as 80% and the level of significance set at <0.05, the preferred sample size was determined as 80. Eighty freshly extracted single-rooted human maxillary anterior teeth free from resorption and structural deformities were collected, cleaned, and stored in distilled water until further use. Teeth were decoronated and sectioned horizontally at the mid-root level to obtain root dentin samples of 2 mm thickness [Figure 1a and b]. The lumen of the root dentin disks was instrumented with Gates Glidden drills (Mani Inc., Tochigi, Japan) of sizes No. 2-5 to achieve a standardized diameter of 1.3 mm [Figure 1c]. All the prepared specimens were randomly divided into two main groups (n = 40) based on the material used to fill the lumen: Group 1– MTA Flow (Ultradent, South Jordan, UT, USA) and Group 2– BD (Septodont, Saint Maur-des-fosses, France). The experimental materials were mixed according to the manufacturer's instructions, as given in Table 1. The mixed material was then introduced incrementally with no pressure into the lumens of the root-dentin disks. A collagen sponge was used as a matrix to prevent extrusion of the material below the inferior surface of the specimens [Figure 1d].

Table 1

Materials used in the study

Material	Manufacturer lot number	Material composition	Manufacturer’s instructions
MTA flow	Ultradent, South Jordan, UT, USA Lot - BKNJF	Powder  Tricalcium silicate  Dicalcium silicate  Bismuth oxide Liquid  Water-soluble silicon-based gel	Powder: liquid ratio of 2:2 (0.26 g powder - 2 drops of gel) and the mix was inserted into the syringe given by the manufacturer
BD	Septodont, Saint-Maur-des-Fosses, Creteil, France Powder - 0459 Liquid - B18644	Powder  Tricalcium silicate  Dicalcium silicate  Calcium carbonate  Iron oxide  Zirconium oxide Liquid  Calcium chloride  Hydro-soluble polymer	The liquid from a single-dose container was emptied into a powder-containing capsule and mixed for 30 s at 4000-4200 rpm in a mixing device

BD: Biodentine, MTA: Mineral trioxide aggregate

Figure 1

(a) Decoronation of sample. (b) 2 mm thick mid-root dentin disk. (c) Instrumentation of root canal lumen using Gates Glidden drill no.5. (d1 and d2 Specimens restored with MTA Flow and Biodentine respectively. (e) subgroups-wrapped in gauze moistened with desired pH. (f) Exertion of load on test material

Each group was divided into four subgroups (n = 10) [Figure 1e] :

Subgroup 1a and 2a– Wrapped in pieces of gauze soaked in PBS at pH 7.4 for 3 days

Subgroup 1b and 2b– Wrapped in pieces of gauze soaked in Butyric acid buffered solution (BABS) at pH 5.4 for 3 days

Subgroup 1c and 2c-Wrapped in pieces of gauze soaked in BABS for 3 days followed by PBS for 30 days

Subgroup 1d and 2d-Wrapped in pieces of gauze soaked in PBS for 33 days.

All the samples were stored in an incubator at 37°C and 100% humidity during the experimental period. The PBS-soaked gauze pieces were replenished daily to ensure a constant pH. After storage, all the specimens were mounted on acrylic blocks with a central hole to allow the free movement of the plunger.

Measurement of push-out bond strength

The samples were positioned on a metal slab attached to the universal testing machine (Instron 8500, Instron Corporation, Canton, OH, USA), and the compressive load was applied by exerting a downward pressure on the surface of the testing material using a 0.75 mm diameter cylindrical stainless-steel plunger at a speed of 1 mm/min [Figure 1f]. A clearance of 0.55 mm was present with the plunger from the margin of the dentinal wall to ensure contact with the test material only. The maximum load applied to the material at dislodgement was recorded in Newtons (N). The POBS values were converted into megapascals (MPa) using the following formula:

Bonded surface area = 2 πr × h

r = radius of root canal (0.65 mm)

h = thickness of dentin slice (2 mm).

Statistical analysis

The results obtained were subjected to statistical analysis using SPSS software version 23.0 for Windows (SPSS Inc., Chicago, IL, USA). Statistical data was presented in the form of mean POBS values [Graph 1]. One-way analysis of variance and Tukey's post hoc test were used for analyzing the data.

Graph 1

Mean push-out bond strength values (MPa) of MTA Flow and BD. *P-value highly significant at 0.01 level

RESULTS

Results showed that all the specimens stored in BABS (pH 5.4) had significantly lower POBS values than those exposed to PBS during the early experimental period. MTA Flow showed the least POBS values when compared to BD. After the acid challenge, BD regained its POBS with aging in PBS (P < 0.01), whereas MTA Flow showed no change in POBS values over time (P > 0.05) [Table 2]. Samples stored in PBS showed the highest POBS in both the experimental periods, of which BD showed the highest POBS compared to MTA flow.

Table 2

Multiple group comparisons using post hoc Tukey’s test

Groups	Multiple comparisons	Mean difference (I-J)	P
Group I MTA flow	PBS (3 days)
	BABS (3 days)	36.37600a	0.000b
	BABS + PBS (3+30 days)	−1.05200	0.947
	PBS (33d)	36.10200	1.000
	BABS (3 days)
	BABS + PBS (3+30 days)	−37.42800	1.000
	PBS (33 days)	−0.27400a	0.001b
	BABS + PBS (3+30 days)
	PBS (33 days)	−37.15400a	0.000b
Group II BD	PBS (3 days)
	BABS (3 days)	31.12000a	0.000b
	BABS + PBS (3+30 days)	−42.14300a	0.000b
	PBS (33 days)	16.50300a	0.000b
	BABS (3 days)
	BABS + PBS (3+30 days)	−73.26300a	0.000b
	PBS (33 days)	−14.61700a	0.000b
	BABS + PBS (3+30 days)
	PBS (33 days)	−58.64600a	0.000b

a Highly significant at 0.01 level, bP value is highly significant at 0.01 level. PBS: Phosphate buffered saline, BABS: Butyric acid buffered solution, BD: Biodentine, MTA: Mineral trioxide aggregate

DISCUSSION

The use of MTA Flow or BD as a retrograde filling material or an apical plug-in cases with pulpal and periapical inflammation result in exposure to a pH of around 5.5 as stated by Lee et al.[2] Evaluating the bond strength of these materials in an acidic environment is of utmost importance before their clinical use as literature showed alteration in microstructure, properties, and setting reaction of these materials. The adhesive strength of these materials is analyzed using the POBS test as it simulates clinical stress by producing pure shear forces.[89] In the present study, the POBS of MTA Flow and BD to root dentin was determined after exposure to an acidic environment (BABS with pH 5.4) during their early setting stages. In clinical conditions, on removing all perpetuating factors causing inflammation, the local tissue pH slowly reverses to neutrality within 3 days.[10] To simulate this healing phenomenon, the samples were exposed to PBS (pH– 7.4) for 30 days after the initial acid challenge for 3 days.

All the specimens were prepared to a standard root canal diameter of 1.3 mm (corresponding to No. 5 Gates Glidden drill) to simulate wide open apices in clinical conditions.[11] BABS was selected because it has been reported to be one of the byproducts of anaerobic bacterial metabolism.[12] PBS with neutral pH similar to tissue fluids was used as a control. A cylindrical plunger of size 0.75 mm was used to rest entirely on the material and deliver forces in a corono-apical direction.

On exposure to BABS, both Group 1b (MTA flow) and Group 2b (BD) showed comparatively lower mean POBS values of 26.245 MPa and 35.039 MPa respectively after 3 days but not significant (P > 0.05). The BABS might have caused surface erosion and dissolution of the formed calcium apatite precipitate resulting in more interfacial gaps and loss of marginal adaptation in both the materials. This is in accordance with Namazikhah et al., who reported the occurrence of porosities in White MTA after soaking in a butyric acid solution that was buffered to a pH of 4.4,5.4 and stated that a high acidic environment leads to more degradation and dissolution of CSBM.[3] Lee et al. noticed the erosion of the cubic crystals with decreased Portlandite peak and loss of needle-like crystals when hydrated at pH 5.[2] Similar erosion might have occurred in BD and MTA Flow, resulting in decreased POBS values in the present study. The needle-like crystals are important in interlocking the entire mass of material, and their disappearance might have caused the material hardness to decrease and solubility to increase when exposed to acidic pH.

BD comparatively showed increased resistance to dislodgment in acidic pH. This could be due to CaCl2 present in the liquid, which penetrates the pores of cement, strongly accelerating the hydration of silicates and leading to their faster crystallization and reducing the setting time, as stated by Thomas et al.[4]

BD could significantly gain POBS on storage in PBS for 30 days after acid challenge (Group 2c >Group 2b). On the contrary, there was no increase in dislodgement resistance of MTA Flow samples (Group 1c similar to Group 1b). The gaps created at the dentin material interface after acidic erosion might have been filled in the BD group due to its continuous release of Ca2+ and OH− ions. These released ions react with phosphate ions from PBS and form uniform, thicker hydroxyapatite precipitates both on the surface and the interface, thus filling the irregularities and resulting in a gap-free interface as suggested by Sarkar et al.[13] Benavides-Garcia M also observed more interfacial gaps with a thinner apatite layer formation in MTA Flow samples compared to BD.[14]

Both MTA Flow and BD showed similar POBS values after 3 days of exposure to PBS (pH 7.4). This could be due to the similarities in their elemental composition and hydration characteristics in PBS. These findings agree with Reyes-Carmona et al., who stated that CSBM interacts with PBS and forms calcium deficient B-type carbonate apatite via an amorphous calcium phosphate phase.[1516]

Mean POBS of BD significantly increased from 66.159 MPa to 108.302 MPa after 33 days of storage in PBS, while MTA Flow showed no change in dislodgement resistance values at three and 33 days. This suggests enhanced adhesion in BD samples to root dentin with time. These findings are alike to those reported by Mustafa et al., who stated that BD undergoes an initial setting within 12 min, forming a hydrated silicate gel and attains the highest compressive strength after maturation for 14 days due to crystallization of the hydrated gel.[17] This bulk set enhances the physicomechanical quality of BD, which might justify the development of higher bond strength values in BD after 33 days. The increase in dislodgement resistance also indicates the superior biomineralizing ability of BD than MTA flow.

The biomineralization ability of CSBM is directly proportional to the amount of Ca2+ ions released by them and the presence of phosphate in the tissue fluids. The higher bond strength values of BD observed in the present study could be attributed to its higher content of calcium-releasing products triggering the formation of tag-like structures at the cement-to-dentin interface, resulting in increased resistance to dislodgement forces when compared to MTA flow.

The hypothesis that explains no significant increase in POBS of MTA Flow after 33 days of aging in PBS might be due to early cessation of the release of remineralizing ions (Ca2+ and OH− ions). This lack of availability of Ca2+ ions might be due to the presence of anti washout gel, which interferes in hydration, as stated by Formosa et al.[6]

The present study being a bond strength analysis, this is only an indirect indicator of the biomineralization ability of the material. Therefore, further studies evaluating the microstructure, interfacial analysis of MTA Flow in comparison to BD in the above-tested conditions should be undertaken and correlated with the results of this in vitro study.

Within the limitations of the present study, it can be concluded that:

Both MTA Flow and BD showed lowered POBS values on exposure to an acidic environment for 3 days

After the acid challenge, BD could regain the bond strength values, whereas MTA Flow showed no change in POBS values on exposure to PBS for 30 days

MTA Flow should be avoided as a retrograde filling material in areas of inflammation and BD should be the material of choice in these conditions.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.