Comparative evaluation of pH and Ca⁺ ion release from MTA on interaction with platelet-rich fibrin and blood clot: an in vitro study

PRF membrane preparation

A 5 mL blood volume was drawn by venipuncture of the antecubital vein from a healthy volunteer and transferred to a 5mL sterile PRF tube (BD vacutainer serum tubes 5mL) without anticoagulant and centrifuged immediately at 3000 revolutions/min (rpm) for 10 min. The resultant PRF clot between the acellular platelet poor plasma and red blood cells was separated out, squeezed on a piece of sterile gauze to obtain PRF membrane.⁸

Sample preparation

Coronal end of the root was sealed with wax (modelling wax, PYRAX). MTA (Angelus Indústria de Produtos Odontológicos S/A, Londrina, Brazil) was mixed (one scoop powder was mixed with 0.33 mL of liquid) and condensed into the prepared canal to obtain a thickness of 4 mm and confirmed by a graduated probe. Fifteen samples thus prepared were divided into three groups.

Group 1 (PRF+MTA)

PRF membrane cut into fragments was placed into the root canal and gently compacted over the MTA using a hand plugger to achieve a thickness of 5 mm.

Group II (BC+MTA)

Blood drawn by venipuncture of the antecubital vein of a healthy volunteer was introduced into the root canal and left to form a clot.⁹

Group III (MTA control)

MTA was mixed according to the manufacturer’s instructions (one scoop of powder was mixed with 0.33 mL of liquid). Cement was then placed into the prepared canal to obtain a thickness of 4 mm and confirmed by a graduated probe.

Experimental model preparation for Ca ion release and pH

The prepared specimens were transferred to sterile falcon tubes containing 10 ml of distilled water⁷ and stored at 37^oC and 100% relative air humidity throughout the testing period. At the end of every experimental period (three hours, seven days and 14 days), the specimens were transferred to a fresh falcon tube containing 10 mL of distilled water and the collected solutions were analysed for pH and Ca⁺ ion release.

Analysis of pH

The digital pH meter (HI5221 Hanna Instruments Cal Check, United States) was calibrated to pH 7 with standard buffer solution before use. The refillable calomel electrode was placed into the falcon tube containing 10 mL of solution to record the pH. The electrode was washed with distilled water and wiped dry between readings.

Analysis of Ca⁺ ion

To determine the release of Ca⁺ ions, ICP-OES Inductively Coupled Plasma- Optical Emission Spectrometry (Thermo Fisher ICAP 7400 ICP-OES, Radial N. American, USA). equipment was used, owing to its high sensitivity and stability for inorganic analysis. It provides rapid and multi-element analysis of the solutions.

Statistical analysis

The pH and Ca⁺ ion release were presented as mean values. One-way ANOVA followed by post hoc Bonferroni test were done to determine the significant differences between groups using IBM SPSS software version 22.0 (IBM Corp., Armonk, NY, USA). The results were considered statistically significant if the p-value was less than 0.05.

pH analysis

The mean pH values of the individual groups at different time periods are presented in Table 1. The control group (MTA) exhibited a mean value of 10.76 at 3h, 11.54 on the seventh day, and 11.16 on the 14^th day, which was significantly higher than the other groups (p=0.000). pH readings for Group 1 (MTA +PRF) were 7.56 at 3h, 7.78 at seven days which increased to 9.10 on the 14^th day. Group 2 (BC) presented pH of 6.68 at 3h which increased to 8.64 on the seventh day and 9.28 on the 14^th day. There was a significant difference in pH between Groups 1 and 2 at 3h and seven days (p < 0.001) but on the 14^th day there was no significant difference between the groups (p > 0.973).

Calcium ion release

The mean Ca⁺ ion release of the individual groups at different time periods is given in Table 2. The mean values of Ca⁺ ions at 3hin the control group (Group 3) was 1.70, MTA +PRF (Group 1) was 0.77 and MTA+ BC (Group2) was 0.72. Group 3 exhibited significantly higher Ca⁺ ion release as compared to Group 1 and Group 2 at 3h. On the seventh day, MTA+PRF (Group 1) had a Ca⁺ ion value of 18.39, MTA+ BC (Group 2) recorded a value of 39.20. The control group (Group 3) recorded a maximum value of 203.08, which was significantly higher than both experimental groups. On the 14^th day Ca⁺ion release from all groups reduced significantly. The recorded Ca⁺ ion release of MTA+PRF (Group 1) was 2.01, MTA+ BC (Group 2) was 2.75 and control group (Group 3) had a value of 4.73. The Ca⁺ ion release from Group 1 and Group 2 was not significantly different for all the experimental time periods. The control group exhibited the maximum Ca⁺ ion release for all the time periods as compared to the experimental groups.

Figure 1. Bar graph representing pH among the groups at different experimental periods.

Figure 2. Bar graph representing Ca⁺ ion release (mg/L) among the groups at different experimental periods.

pH

An alkaline environment promotes osteogenic differentiation and bone formation. Mineralisation enzymes such as alkaline phosphatase peaks at pH 7.37 and significantly diminished under physiologic level. In vitro and in vivo research has shown that a pH above 8.0 inhibits the mineralization process. Therefore, various biological and molecular responses that influence repair and regeneration may depend on the local pH.

It is important to create an antibacterial environment at the tooth restoration interface or to control the residual bacteria in the canal space to reduce the risk of reinfection.¹⁸

White MTA (WMTA) when in contact with tissue fluid, MTA dissolves and releases hydroxyl ions (OH^-) increasing the pH to 11-12 and contributing to reparative dentin formation.¹⁹ Hydroxyl ions released from biomaterials also create a hostile environment for bacterial survival and proliferation reducing reinfection. Longer exposure to an alkaline environment could have induced potential degradation and a subsequent decrease in the detection of the growth factors.²⁰

All experimental groups showed an increase in pH over period of time. The highest alkaline pH was recorded in the control group, MTA with distilled water at seven days (11.54) and the lowest in PRF/MTA group (7.78). The difference in pH between PRF and blood clot at 3h and seven days (p < 0.001) was significant, but on the 14^th day these groups recorded similar values. Group 3 recorded a mean pH value of 10.76 at 3h, 11.54 on the seventh day and 11.14 on the 14^th day which was significantly different from the experimental groups. This is in accordance with the study.⁹ In the same study, WMTA with blood recorded higher pH values in contrast to our study where the BC with MTA group recorded lower pH values. This could be because the experimental set up was different in that study: in that study, the MTA cylinder was prepared and immersed in blood whereas in our study, PRF/BC was placed over MTA and the pH recorded.

Calcium ions

Calcium ions are known to act on osteoblasts and cementoblast cells, causing their differentiation and hard tissue mineralization. Hunter et al. (2018) reported that calcium ions released from biomaterials may influence the repair process, as they pass through the cell membranes by depolarization or activation of membrane-bound calcium channels.²¹ Although Ca⁺ ions are one of the major components released by MTA, the role of Ca ions in regenerative endodontics is largely underexplored. In the present study, the mean values for Ca⁺ ion release from Group1 (PRF/MTA) and Group 2 (BC/MTA) was 0.77 mg L^-1 and 0.72 mg L^-1 respectively at 3h, which were not significantly different from each other. At seven days, Group 1 recorded a value of 18.39 mg L^-1 and Group 2 recorded a value of 39.20 mg L^-1. Though the mean Ca⁺ ion release values of Group 2 were higher than for Group 1, it was not statistically significant at seven days (p ≤ 0.001). At day 14, there was significant reduction in the Ca⁺ ion release where Group 1 recorded a value of 2.01 mg L^-1 and Group 2 recorded a value of 2.75 mg L^-1, which were again not significantly different from each other. Overall Group1 (PRF/MTA) showed the lowest release of calcium ions compared to Group 2 (BC/MTA) in14 days. At all experimental periods, Group 3 recorded the maximum Ca⁺ ion release, which was statistically significant for both experimental groups. A previous study⁹ evaluated Ca⁺ ion release from WMTA with and without blood respectively, and reported that the group with WMTA and blood showed greater calcium ion release, which is contrary to what was recorded in our study. This could be because the experimental set up was different in that study, where a MTA cylinder was prepared and immersed in blood; in contrast, in our study, blood clot was placed over MTA and the ion release was recorded. To measure the release of Ca⁺ ions, ICP-OES equipment was used, owing to its high sensitivity and stability for inorganic analysis. It provides rapid and multi-element analysis of the solutions. In this in vitro study, both experimental groups showed diffusion of Ca⁺ ions from MTA through scaffolds at three hours, seven days and 14 days, though PRF/MTA allowed lower diffusion of Ca⁺ ion as compared to the blood clot. However, various clinical studies have suggested that PRFs induce regeneration of pulp dentine complex better than blood clot as it is a rich source of platelets and growth factors.¹⁷

There have been a limited number of studies evaluating the effect of PRF/BC with MTA on the pH and calcium ion release. In the present study, we observed that PRF and BC influenced the pH and Ca⁺ ion release from MTA.