Bacterial Leakage Assessment for Different Types of Resin-Based Dental Restorations Applied Using Various Placement Methods

Introduction

Composite resins are the materials of choice for most restorations in today’s clinical dentistry (1) because of their biocompatibility and absence of mercury, ability to match tooth color, thermal non-conductivity and ability to bond to tooth structures (2,3). Although composite resins have improved greatly since their introduction, polymerization shrinkage of 1.5-5% and microleakage as a result, are the most frequently encountered problems (4). Microleakage is defined as the passage of fluids, bacteria or molecules between the cavity walls and restorative materials (5). Microleakage may lead to postoperative sensitivity, enamel fracture, marginal staining, recurrent caries, eventual failure of restorations, and the development of pulpal pathology (6). Although the protective functions of dentin and the capacity of the pulp to sustain bacterial challenges have been demonstrated (7), the defense mechanisms of the dentin/pulp complex must not be put to the test. Therefore, bacterial leakage at restoration margins is a major concern. To minimize volumetric shrinkage, efforts have been directed toward slowing down the composite resin polymerization rate (8), using an incremental placement technique (9), placing thicker adhesive layers under the composite (10) or use of low-modulus intermediate layers (11). Various studies have also reported efforts to develop a non-shrinking high-performance polymer for use as a matrix material for dental composite resins (12). Siloranes, a new category of ring-opening monomers, were introduced to overcome the problems associated with volumetric shrinkage. The volumetric shrinkage of silorane-based composite resins is less than 1% (13), which is due to opening and extending the oxirane rings during polymerization, compensating for volume reduction (13,14). The use of flowable composite resins as liners has been suggested to improve adaptation to cavity walls, reducing microleakage. These effects may be due to its low viscosity, increased elasticity and wettability (15). Various techniques have been used in microleakage studies. Bacterial penetration test is a non-destructive technique that was originally described to test bacterial leakage around filling materials (16), and later it found applications in endodontics (17). Since there is a clear relationship between dentin infection, pulp inflammation and loss of pulp vitality, the question regarding possible differences in capacity of different restorative materials to prevent bacterial microleakage is very important. The aim of this study was to compare the effects of different resin-based dental restorations and layering techniques on bacterial microleakage of Cl I composite resin restorations.

Materials and Methods

This in vitro study was carried out on 82 newly erupted non-carious, non-restored, human third molars, gathered following informed consent, approved by the Commission for Medical Ethics of Mashhad University of Medical Sciences (N0. 900672). The teeth were disinfected by storage in 0.02% thymol solution for 24 hours and stored in normal saline solution until use (18). The occlusal enamel was trimmed at the level of the main grooves using a slow-speed disc (KG Sorensen, Barueri, SP-Brazil) under copious running water, exposing an occlusal flat enamel surface. Uniform box-shaped Class I cavities were prepared measuring approximately 4 mm (mesial-distal) × 4 mm (buccal-lingual) × 2 mm (in depth) at the occlusal crown center, using a high-speed handpiece with fissure burs # 109/008 (Brasseler, Savannah, GA, USA) under constant water irrigation for all the cavities. Cavity dimensions were measured by a periodontal probe. The burs were changed every five preparations. The cavosurface margins were prepared at 90°. All the prepared Cl I cavities consisted of enamel and dentinal walls and the pulpal floor was located on dentin. These dimensions yielded a box-shaped cavity with a C-factor of 4 (bonded surface/unbonded surface area = 64 mm²/16 mm² = 4).

The prepared teeth were then randomly divided into two main groups of 36 teeth each and two positive and negative control groups of five teeth each as follows.

Group 1: a silorane-based composite resin (Filtek P90, 3M ESPE)

Group 2: a methacrylate-based composite resin (Clearfil AP-X, Kurary, Japan)

Either FiltekSilorane System Adhesive with Filtek P90 composite resin or Clearfil SE Bond with Clearfil AP-X composite resin was applied to the cavity as follows:

The Filtek P90 primer was applied to the entire cavity wall and left over the entire area for 15 sec. A gentle stream of air was used and the primer was cured for 10 seconds. The bonding agent was applied to the entire cavity walls and cured for 10 sec.

The Clearfil SE primer was applied to the entire cavity wall and left in place for 20 seconds.

Then, the volatile ingredients evaporated with a mild air stream. The bonding agent was applied to the entire cavity walls and cured for 10 seconds.

The adhesives were light-polymerized using an Optilux 501 quartz tungsten halogen unit (Demetron‒Kerr, Orange, CA, USA) set at 600 mW/cm². All the bonding procedures were carried out by a single operator at a room temperature of 24°C. Each of the two main groups was subdivided into three subgroups A, B and C (n = 12) based on three different placement techniques:

Subgroup A (bulk filling): Shade A₂ composite resin was placed in one bulk and cured for 40 seconds.

Subgroup B (incremental filling): Shade A₂ composite resin build-ups were constructed in two 1-mm-thick horizontal increments, which were individually light-polymerized for 20 seconds.

Subgroup C (snowplow): The first layer consisted of 0.5-mm-thick Filtek Z 350 flowable composite resin. Then shade A₂ composite resin was condensed over the uncured flowable composite resin; excess flowable composite resin was removed with an explorer and then light-cured for 20 seconds. The rest of the cavity was filled similar to that in subgroup B. After finishing the restorations, additional curing of occlusal aspects of each tooth was carried out for 40 seconds. Then the teeth were finished and polished with rubber cups and points (Identoflex, Kerr Hawe SA, Bioggio, Switzerland).

Positive control group: The five teeth used in this group were left empty after cavity preparation to provide a passage for bacterial leakage.

Negative control group: In this group (n=5), sound teeth were used and all the surfaces were coated with two layers of nail varnish to prevent bacterial leakage.

All the restored specimens were stored for 24 hours in distilled water at 37°C and subjected to 1000 thermal cycles at 5°C/55°C with a 30-second dwell time.

Bacterial Leakage Assessment

Following the removal of the roots, the dentin between the furcation and the pulp chamber floor was also removed. The external surfaces of all the specimens, except for 2 mm around the restoration, were covered with two layers of nail varnish. The microbial test consisted of a 2-chamber method with some modifications (16). Each specimen was embedded in one end of a plastic tube with epoxy resin (Meliodent, Heraeus-Kulzer, Germany). The junctions between the crown, epoxy resin and the tube were sealed with cyanoacrylate adhesive. The mounts were sterilized for 8 hours in an ethylene oxide sterilizer (Anprolene, AN 74C, Andersen Products Inc., Haw River, NC, USA) at room temperature. After sterilization, the apparatus was placed in a glass flask (the lower chamber) containing sterile brain-heart infusion broth (BHI, ScharlauChemie S.A., Barcelona, Spain). 2-3 mm of the specimens was immersed in the broth. The junctions between the plastic tubes and the glass flasks were tightly sealed with Parafilm and cyanoacrylate adhesive. An initial bacterial suspension containing 1.5×10⁸ CFU/mL of S. mutans (ATCC 25175) was used. The upper chambers were filled with 8 mL of the initial suspension keeping the bacterial suspension in contact with the occlusal surface of the specimens (Fig 1). The mounts were always handled in sterile conditions under a laminar flow hood (Nuaire, Plymouth, MN, USA) to avoid bacterial contamination. They were placed in an incubator at 37ºC for 3 days. The lower chambers of all the mounts were observed daily and the turbidity time was recorded for each specimen. Once turbidity was present, a sample of the turbid broth was streaked onto blood agar plates and the bacteria were identified to ensure that there was no contamination other than S. mutans. The results were analyzed using Fisher’s exact and chi-squared tests. Statistical significance was set at P<0.05.

Figure 1. The assembly used for testing bacterial leakage.

Results

Complete leakage was recorded in positive controls, while negative samples showed no leakage during the experiment. The results of bacterial leakage are shown in Tables 2 and 3.

Statistical analysis revealed no significant differences between Filtek P90 and Clearfil AP-X (P=1) in terms of microleakage. The differences between the results, which were related to different filling techniques, were not significant, either (P>0.05).

Table 1. Materials, chemical compositions and application procedures

Table 2. Comparison of bacterial microleakage in different subgroups of thetested materials

Table 3. Comparison of bacterial microleakage in two resin composites