Effect of blood contamination and various hemostatic procedures on the push-out bond strength of Biodentine when used for furcation perforation repair.

BACKGROUND: Perforations in the furcation area are common procedural accidents that can impact the outcome of treatment. There are many bioactive materials available to repair these defects. AIM: The purpose of this study was to determine and compare the effect of 25% aluminum chloride solution, 20% ferric sulfate solution, and a 980-nm diode laser, when used for hemostasis, on the dislocation resistance of Biodentine placed to repair furcation perforation.
MATERIALS AND METHODS: This in vitro study was conducted on fifty extracted human permanent mandibular molars, with ten teeth in each group. The stimulated perforations were contaminated with blood, except for one group. The contaminated groups were either treated with aluminum chloride, ferric sulfate, diode laser, or none at all. All the perforations were restored with Biodentine and tested for push-out bond strength. STATISTICAL ANALYSIS USED: One-way analysis of variance and Tukey's HSD post hoc test were applied with a level of significance set at 0.05.
RESULTS: The dislocation resistance of Biodentine was found to be highest when aluminum chloride or diode laser was used for arresting bleeding. In contrast, the ferric sulfate group gave the lowest value for push-out bond strength (P < 0.05).
CONCLUSIONS: According to the present study, the use of ferric sulfate as a hemostatic agent showed a negative effect on the bond strength of the calcium silicate cement to dentin. Furthermore, Biodentine performed better when diode laser and aluminum chloride were used for hemostasis. Copyright:

INTRODUCTION

Furcation perforations are common procedural complications that occur during root canal therapy or preparation of post space due to iatrogenic intervention or caries. These perforations must be repaired immediately. Biodentine (Septodont, Saint-Maur-des-Fosses, France), a tricalcium silicate-based hydraulic cement, has been recommended as a dentin substitute because of its ability to seal, short setting time, greater compressive strength, and bio-induction properties. A significant concern during perforation repair and sealing is uncontrollable hemorrhage. It is shown to interfere with the sealing ability and bond strength of calcium silicate cement.[12] Therefore, to circumvent these concerns, various hemostatic agents can be used for easier fluid control and blood coagulation.

Aluminum chloride and ferric sulfate are traditionally used as astringents. Another method that can be considered for hemostasis is the use of diode laser. It is considered a minimally invasive technology that offers greater advantages, superior to those of the conventional hemostatic agents with regard to being biocompatible.[3] The purpose of this study was therefore to determine and compare the effects of diode laser, ferric sulfate, and aluminum chloride as hemostatic agents on Biodentine by measuring the push-out bond strength when used as a furcal perforation repair material.

MATERIALS AND METHODS

Experimental design

This was a double-blinded (technician and statistician) in vitro study, conducted after obtaining Institutional Ethical Committee approval (reference number 19057).

Sample selection

Human extracted permanent mandibular first and second molars considered for the study were cleaned using an ultrasonic scaler to remove surface debris and were stored in distilled water until use. The sample size was calculated based on a study that determined the effect of root dentin conditioning on the push-out bond strength of Biodentine.[4] The number of samples required per group with a power of 99% and an alpha error rate of 1% was 7. For efficient calculation and accounting for errors in testing, a sample size of 10 per group was finalized. Fifty teeth required for the study had no caries extending especially to the cervical third, no fused roots, no previous endodontic treatment, and no craze lines/fracture extending toward the cementoenamel junction when observed under ×7 magnification (Seiler IQ Dental Microscope, St. Louis, Missouri, USA).

Specimen preparation

All these samples were marked with a pencil at 4 mm above the pulpal floor and 4 mm below the furcation area, with the help of a periodontal probe. The teeth were decoronated till the markings above the floor of the pulp and roots were amputated below the furcation area at the marking point using a diamond disc (Strauss Diamond, 321-Flex Double Sided disc, 0.20 mm thickness Palm Coast, FL, USA) at slow speed. A furcal perforation was prepared with an approximate depth of 3 mm and diameter of 1.3 mm using #4 Peeso Reamers (Mani, Tochigi, Japan). The samples were then mounted in polymer tubes of dimensions 3 cm × 3 cm using cold cure acrylic by blocking the perforation site with Teflon tape. The prepared models were mounted in such a way that at least 2 mm of space was seen between the furcation area and acrylic block.

Grouping

These prepared samples were then randomly allocated into five groups, with ten samples per group (n = 10). Fresh blood collected using a 5-ml syringe from the principal investigator was used for contaminating the perforations. Detailed information about the materials used in the study is shown in Table 1.

Table 1

Materials used with composition, manipulation, and mode of application

Materials	Details	Manipulation/manufacturer instructions
Biodentine (Septodont, Saint-Maur-des-Fosses, France)	Powder: Tricalcium silicate and dicalcium silicate (core material), calcium carbonate (filler), zirconium oxide (radiopacifier)Liquid: Calcium chloride, hydrosoluble polymer	Five drops of liquid from a single-dose container dispensed into the capsule. It is then mixed in an amalgamator for 30 s at 4000 rpm
Hemostal (Prevest, DenPro, India)	25% buffered aluminum chloride, glycerine, purified water, and excipients	Applied directly to the required place with saturated cotton pellet for 3 min
Hemostaz (Maarc Dental, India)	Ferric sulfate 20%, liquid equivalent with insert binding agents in a liquid having a pH of ~1.0	Applied firmly for 3 min till there is a color change with clot formation
Diode laser (Wiser, Doctor Smile, Italy)	Wavelength: 980nmPower: 0.8 wattMode: Continuous waveFiber tip diameter: 300 microns	Lasing was done in small circular movements over the area with slight contact for 10 s for each tooth

Group 1 (blood contamination group without hemostatic agents; blood): In this group, freshly collected blood was taken in a 27-gauge needle and a drop of blood was injected into the perforation cavity. The excess blood was removed using 10-s irrigation with saline, and the defect was dried with paper points (Dentsply Tulsa Dental, USA, size: 80; taper: 0.02).

Group 2 (blood contamination group coagulated with aluminum chloride; AlCl3): In this group, a drop of freshly collected blood was injected into the perforation. The blood was coagulated by placing cotton pellets dipped in 25% aluminum chloride for about 3 min. The perforation was then irrigated with saline for approximately 10 s and then dried using paper points.

Group 3 (blood contamination group coagulated with ferric sulfate; FeSO4): In this group, freshly drawn blood was taken in a 27-gauge needle and a drop of blood was injected into the prepared cavity. Blood was coagulated by placing cotton pellets dipped in 20% ferric sulfate for about 3 min, washed with saline for 10 s, and was dried with paper points.

Group 4 (blood contamination coagulated with diode laser; laser): In this group, a fresh blood was injected into the perforation. The blood was coagulated using a 980-nm diode laser (Wiser, Doctor Smile, Italy) of 0.8 W for about 10 s and was washed saline and dried.

Group 5 (control group with no blood contamination; saline): In this group, the furcal perforation created was irrigated only by saline for 10 s and dried with paper point.

Placement of repair material

After the simulated furcal perforations were treated with the respective solutions, Biodentine (Septodont, Saint-Maur-des-Fosses, France) was mixed and placed as per the manufacturer's instructions [Table 1]. A cotton pellet moistened with water was placed over the material for 24 hours.

Measuring push-out bond strength

Push-out bond strength was carried out using a universal testing machine (Zwick/Roell, Kennesaw, Georgia, USA) at crosshead speed of 1 mm/min in occluso-gingival direction and parallel to the long axis of the tooth. The Biodentine placed in was pushed using a cylindrical metal plunger with a tip diameter of 1.0 mm until cement was dislodged. The maximum load to dislodge the cement was measured in Newtons (N) and converted to megapascal (MPa).

The push-out bond strength values obtained in N were converted into MPa by using the following formula:

Bond strength (MPa) = Dislodgment force (N)/surface area bonded (mm2)

Surface area bonded = Perforated area diameter ×π× perforation height.

Statistical analysis

The values obtained were tabulated and subjected to statistical analysis using SPSS (version 20, SPSS, Inc., Chicago, IL, USA). The outliers were identified using a Q–Q plot. These were deleted from the final analysis as it could affect the result. The data were found to be normally distributed as suggested by Shapiro–Wilk test. Intergroup and intragroup comparison was carried using one-way analysis of variance and Tukey's HSD post hoc test with P value set at 0.05.

RESULTS

The push-out bond strength values for each group are depicted in Figure 1. The least push-out bond strength was noticed when perforation was exposed with ferric sulfate (6.10 ± 4.68 MPa). The highest push-out bond strength was observed on exposure with aluminum chloride (13.26 ± 3.22 MPa). Only blood contamination (12.55 ± 3.92 MPa) and hemostatic procedure with laser (12.02 ± 6.16 MPa) had shown similar bond strength values.

Figure 1

Descriptive statistics of push-out bond strength (MPa) of each group (*Different alphabets next to the values indicate significant difference)

There was no statically significant difference in the bond strength between the control groups and the group treated with either aluminum chloride or laser. However, the bond strength of Biodentine significantly reduced in the ferric sulfate group when compared to the samples after blood contamination and those treated with aluminum chloride (P < 0.05). The details of post hoc analysis are given in Table 2.

Table 2

Intergroup comparison of the push-out bond strength

	Blood	AlCl3	FeSO4	Laser	Saline
Group 1 blood
Mean difference	-	−0.700	6.45	0.536	4.96
P	-	0.997	0.036*	0.999	0.163
Group 2 AlCl3
Mean difference		-	7.15	1.236	5.661
P		-	0.016*	0.978	0.084
Group 3 FeSO4
Mean difference			-	−5.917	−1.49
P			-	0.064	0.956
Group 4 laser
Mean difference				-	4.425
P				-	0.257
Group 5 saline
Mean difference					-
P					-

*Statistically significant difference

DISCUSSION

Iatrogenic furcal perforation during posterior endodontic access preparation is a frequent mishap with a guarded prognosis due to its approximation to the gingival sulcus. On occasions, extensive carious lesions can also lead to furcation perforation.[5] Once this untoward event occurs, every effort should be directed to seal the perforation immediately with a biocompatible, bioactive material that can prevent microleakage and withstand dislodgement forces. In some instances, when there is a delay in repairing the defect, granulation tissue can invade the lesion. This can result in excessive hemorrhage which necessitates the need for hemostatic agents/procedures to avoid the erosion of the repair cement.

The choice of material in the present study was Biodentine a calcium silicate cement with superior properties. Biodentine demonstrated bonding to dentin through mineral infiltration zone into the intertubular dentin as well as tag-like structures into the dentin tubules.[67] It has increased push-out bond strength, better compressive strength, greater density, decreased porosity, color stability, induction of cell proliferation and biomineralization, low cytotoxicity, gingival fibroblast viability preservation, and faster setting time (12 min) with wide clinical applicability.[89] Any chemical or morphological alteration of dentin can affect the interfacial interaction of the calcium silicate cement to dentin.[7] With the various effects of contaminants on the resistance to repair material, dislodgment is an important determinant for the long-term prognosis. Therefore, this study was conducted to assess the dislocation resistance of Biodentine to dentin after subjecting it to subject to various hemostatic procedures.

In the present study, push-out bond strength testing was carried out to determine the dislodgment resistance of the repair material. This technique is shown to be a reliable method for evaluating the bond between the perforation repair materials with the dentinal wall.[10] In order to simulate the clinical scenario better in the current study, the perforation in the furcal region was contaminated with blood in all the groups, except the control group. The bond strength of Biodentine to the uncontaminated control group was very low (7.59 ± 2.40 MPa) and unexpectedly the Biodentine bond strength to the blood-contaminated group was much higher, though not statistically significant (12.55 ± 3.92 MPa). This is in agreement with the research by Singla et al.[11] which revealed that the bond strength of Biodentine on contamination with blood was higher when compared to other cement. The reason could be due to the formation of hydroxyapatite crystals at the surface on interaction with the blood. The apatite crystals might have the potential to increase the ability in sealing, especially when formed at the material interface with dentinal walls which could explain the increased bond strength of Biodentine. In the research done by Aggarwal et al.,[1] the bond strength of Biodentine had shown no significant effect of blood contamination when used as a furcal perforation sealing material. The results of the current study are contrary to the experiment by Shalabi et al.[12] in which contamination of blood during Biodentine setting gave significantly poor push-out bond strength results. They had used Biodentine as a root-end filling to the apical end of radicular dentin, and the unset root-end filling material was placed into Eppendorf tubes containing blood.

In the present study, the effect of two commonly used hemostatic agents (aluminum chloride and ferric sulfate) on the dislocation resistance of Biodentine was assessed. The group where 25% buffered aluminum chloride (AlCl3) was used as a styptic demonstrated the highest bond strength (13.26 ± 3.22 MPa). The mechanism by which AlCl3 stops bleeding involves the coagulation of proteins in the blood, thereby preventing the outflow of blood from the vessels.[13] The increase in bond strength after applying AlCl3 may be explained by the formation of aluminum hydroxyapatite crystals as suggested by previous studies.[1314]

On the contrary, the experimental group in which 20% ferric sulfate was used showed the lowest bond strength (6.10 ± 4.68 MPa). Bernades Kde et al.,[15] in their systematic review, had described that due to its acidic nature, morphological changes were observed in dental hard tissue, which might be one of the reasons for reduced push-out bond strength of Biodentine when ferric sulfate was applied. The astringent solutions mentioned above can cause gingival tissue irritation and have been shown to impair bone formation.[16] Hence as an alternative, diode lasers can be considered in achieving hemostasis.

The 980-nm diode laser group had given higher bond strength values (12.02 ± 6.16 MPa), almost equal to the aluminum chloride group. These results agree with the study by Mohammadian et al.,[17] where the dislocation resistance of calcium silicate cement to dentin when conditioned with 980-nm diode was increased. The probable reason for this could be due to the removal of the smear layer from the dentin surface and penetration of cement into open dentinal tubules.[18] The lower power setting used in this study would have prevented the melting of dentin surface and sealing of tubules, thus improving the formation of tag-like structures into the dentinal tubules.[1920] Among the various biologic effects of laser on tissues, the principle of photothermal effect is mainly employed. Photothermal effects include coagulation, soft-tissue incisions, granulation tissue removal, hard tissue incisions, and ablation. Laser energy absorption depends on the wavelength of the laser and the optical properties of the tissues. Diode lasers 980 nm have a detrimental effect on hard tissues. Although tissue perforation is less when compared to other soft-tissue lasers, it causes ablation and damages root cementum and bone. Lower energy settings such as <1 W coupled with shorter exposure time, in agreement with the present study, do not cause irreversible damage.[2021] There is, however, a need for future studies to evaluate the effect of various hemostatic agents and procedures on radicular dentin properties and to determine how they affect other hydraulic endodontic repair cement.

CONCLUSIONS

The push-out bond strength of Biodentine was found to be highest with the aluminum chloride group followed by the laser group and the ferric sulfate group. The bond strength of Biodentine was negatively affected by ferric sulfate. Based on the results of this study, diode laser and aluminum chloride are preferred for arresting bleeding. As diode lasers have similar results to aluminum chloride, they are an excellent choice for coagulation, as they are said to have less adverse effects on tissues than other hemostatic methods.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.