RESULTS

Statistical analysis

Data was entered into Microsoft excel sheet and analysed using SPSS version 21 (IBM). The outcome of the study

(presence of leakage) was categorical in nature. Intergroup comparison of frequency of leakage was done using Chi square test. The level of statistical significance was set at 0.05. The graphs were prepared on Microsoft Excel (Table 1). Results show that all the root end filling materials exhibited microleakage. None of the negative control groups showed any color change indicating no leakage and all of the  positive controls demonstrated a color change indicating leakage within the first 24 h. The numbers of samples of experimental groups with bacterial leakage at the end of 1^st, 2^nd, 3^rd and 4^th week are listed in Graph 1. 

The difference between the frequencies of leakage among all the four study groups was found to be statistically significant (p>0.05) at 1^st week, 2^nd week and 3^rd week but it was non- significant at the end of 4^th week.

On inter group comparison, difference between group 1 and group 3 was statistically significant at 1^st week, 2^nd week and 3^rd week. While between group 1 and 4 difference between the frequencies of leakage was significant at 1^st week and 2^nd week. On comparison between group 2 and 3, this difference was significant only at the end of 1^st week. At the end of 4^th week, there was no significant difference in any of the groups regarding frequency of microleakage.

DISCUSSION

The purpose of placing a retrograde seal after apicoectomy is to establish an effective barrier between the root canal and the periapical tissues. The apical seal is the single most important factor in achieving success in surgical endodontic. A plethora of materials have been suggested for use as root end filling and have been tested for their sealing ability. Among the various root end filling materials tested, Mineral Trioxide Aggregate (MTA) has shown good sealing ability and biocompatibility in previous in vitro and in vivo studies [13]. The powder particles are hydrophilic and small. When they come in contact with the moisture the  hydration reaction occurs that results in colloidal gel structure which solidifies in the mineralized tooth structure [14]. The main characteristic of white MTA was the near-elimination of iron from the original formulation, depleting the set MTA of aluminoferrite, which was responsible for the gray coloration. This color was problematic in some circumstances where the cosmetic appearance of the treated tooth was affected adversely. Biodentine is a new calcium silicate-based root end filling material which uses novel active biosilicate technology to ensure superior mechanical properties [14]. In addition to the chemical composition based on the Ca₃SiO₅ and water chemistry which brings the high biocompatibility of already known endodontic repair cements like MTA, it has increased physico-chemical properties like short setting time, high mechanical strength which make it easy to handle and compatible, not only with classical endodontic procedures, but also for restorative cases of dentine replacement [15]. The search for alternative materials is aimed to reduce costs and to increase the feasibility to both professional and patient. PMMA is a new material that might potentially provide the necessary properties of a root end filling material. PMMA bone cement has been widely used in orthopedic surgery [7].

As there is limited literature for PMMA as a retrograde filling material the aim of this study was to compare its sealing ability with gray MTA, white MTA and biodentine as a root end filling material by using bacterial leakage model.

A successful periapical surgery requires appropriate root-end resection, preparation and adequate apical seal. Apical root- end resection of 3 mm at 90° angulation was done in our study to reduce 98% of the apical ramifications and 93% of the lateral canals, which might be responsible for endodontic failure [16]. 90° plane of sectioning was selected for this study as it has been proved to be most acceptable by earlier studies. The inclined plane sectioning at 30 or 45° angle could have disadvantages like open dentinal tubules, errors in post-operative radiographs, more mechanical stresses, loss of dentin, cementum and bone that could result in compromised healing [15].

Traditionally, the root-end cavity is prepared with burs used with low-speed hand piece. Cavity preparation with burs has some disadvantages including limited operative field and root-end bevel, which increases the number of exposed dentinal tubules on the root-end surface. In the present study, ultrasonic surgical tips were used as an alternative to the burs for root-end cavity preparation. It allows cleaner and deeper cavity centered in the root canal and reduced bevel angle thus having the advantage of decreased number of exposed dentinal tubules at the resected surface and so the microleakage is also minimized [17].

In literature, several in vitro methods have been used to assess the sealing ability of root-end materials: dye penetration, radioisotopes, bacterial leakage studies, electrochemical techniques, scanning electron microscopy, and fluid filtration methods [1]. Among these techniques the bacterial leakage model seems to be the most clinically relevant method [6]. Thus, it was used in the present study to evaluate microleakage in vitro. It has been argued in the case of dyes, if the size of the dye molecules is small enough, a false positive result may be obtained. In contrast, bacteria would be a better tracer, especially when testing leakage of hydrophilic materials. Moreover leakage tests involving microbiology are more technically exacting. The bacterium selected as the leakage marker should preferably be an endodontic pathogen, and easy to cultivate [18]. Enterococcus fecalis was chosen as the bacteriological marker in this study It is commonly found in a high percentage of root canal failures and is able to survive in the root canal as a single organism or as a major component of the flora, while other bacteria commonly associated with endodontic infections may require symbiotic support from other bacteria [2,12]. Under these experimental conditions, all the root end filling materials exhibited microleakage. PMMA samples exhibited least microleakage (26.7%) followed by biodentine (40.0%), gray MTA (46.7%) and white MTA (53.3%) after 4 weeks.  It was noted that biodentine exhibited least microleakage up to 3 weeks but at the end of 4^th week PMMA showed least microleakage. Although the difference in results seems to be clinically relevant but they did not reach the level of statistical significance. Though statistically not significant, gray MTA showed less bacterial micro-leakage in comparison to white MTA in the present study. Aranha et al. [19], Matt et al. [20], Al- Hezaimi et al. [21] and Ferris and Baumgartner [22] showed more leakage with WMTA compared with GMTA, although the differences between the two materials in these studies were not statistically significant, perhaps the small differences were caused by differences in setting expansion of GMTA and WMTA. Kazem et al. [2] found no significant difference between gray MTA and white MTA in terms of bacterial leakage. Their results are consistent with the present study. In the present study, the sealing ability of biodentine was found better than both gray and white MTA in early stages. Findings of our study were consistent with the results of Ravichandra et al. [12] and Kokate and Pawar [15]. A study conducted by Han and Okiji [23] demonstrated that Biodentine has more prominent biomineralization ability than MTA, with wider calcium and silicon rich layer at material-dentine interface. Soundappan et al. [16] and Mandava et al. [14] observed different results in their respective studies which showed that the microleakage of MTA was comparatively less than Biodentine. The disparity in results of these two studies may be attributable to differences in the methodology employed. Soundappan et al evaluated the marginal adaptation of Biodentine in comparison with Mineral Trioxide Aggregate (MTA) as a root end filling material, using Scanning electron microscopy (SEM) and Mandava et al. [14] evaluated the apical microleakage of Mineral trioxide aggregate and Biodentine using dye penetration under stereomicroscope whereas in the present study, sealing ability of Biodentine and MTA was evaluated using bacterial leakage model. In the present study PMMA bone cement has shown the better sealing ability than white MTA in the early stages while it showed comparable results with gray MTA, white MTA (later stage) and biodentine. Similar study by Girish et al. [7] comparing PMMA bone cement with other root end filling materials showed that PMMA bone cement exhibited less microleakage when compared with calcium phosphate cement and MTA. The current study is an attempt to evaluate the use of polymethylmethacrylate bone cement in the field of endodontic where the search for the ideal repair material is a continuing process. Further research is required to assess the bioactive property of the bone cement especially the longevity of calcium release and long term stability in relation to body tissues and tissue fluids after its placement.

CONCLUSION 

Within the limitations of this study, it can be concluded that the seal provided by PMMA bone cement as a root end filling material was comparable to MTA and biodentine. It had better working properties, which could overcome potential disadvantages faced with MTA. PMMA bone cement thus seems to be an excellent and promising economical material for root end filling.