Biocompatibility of Newly Prepared Nanocalcium Oxide Based Root Canal Sealer (In Vivo study)

,


INTRODUCTION
Biocompatibility is an essential requirement of any root canal sealer; as the root filling material constitutes a true implant coming into direct contact with the vital tissue at the apical and lateral foramina of the root (1,2) .The sealer is said to be biocompatible, when it comes into contact with the periapical tissue; fails to trigger an adverse reaction such as toxicity, irritation, inflammation or necrosis and it can be permitted or induced periapical tissues healing and repair (3,4) .
The chemical composition of the endodontic sealer may influence positively or negatively on the final result of the endodontic therapy.Therefore, it must be nonirritating and biocompatible with the living connective tissues (5,6) .
Bioceramic sealers have been brought into the scope of endodontic sealers due to the following properties: high pH, excellent seal (bond chemically and micromechanically to dentin), biocompatible, bioactive, and permit healing of the surrounding periapical tissue (7,8)   .
Many researchers reported that the production of sealers with nanosize level can improve their physicochemical characteristics, increasing the biocompatibility and biomineralization abilities, enhancing their antibacterial property, and provide good sealing ability (9,10)   .
Biocompatibility of endodontic materials is usually evaluated by in vitro cytotoxicity using cell culturing methods or in vivo implantation (subcutaneous and/or intraosseous) using laboratory animals.However, although the cell culturing methods give some valuable information about the response of specific cells to the tested material, they do not provide the full picture of how a tissue reacts to the material and cannot reflect the healing reactions of living tissue under in vivo conditions (11,12) .
The purpose of this study was to evaluate the reaction of the subcutaneous connective tissue to a newly prepared sealer.

Nanosealer
The sealer consists of powder part and liquid part; after several pilot studies, the final formula for the prepared sealer that gave the best clinical sealer consistency and properties within the limits of ANSI/ADA Specification No.57/2012 (13) was as the following.The powder part consists mainly of nano CaO (44%) in addition to Glutamic amino acid, zirconium oxide (20nm), and silica oxide (15-20)nm, while the liquid part consists mainly from distilled water (82%) in addition to propylene glycol.The powder/liquid ratio was 1g powder part to 0.3ml liquid part and the mixing time was

Biocompatibility Test
The biocompatibilty study was conducted according to the ISO 10993-6 (14) (Biological evaluation of medical devices: Tests for local effects after implantation).

Groups Comparison Histopathologically
Histopathological representative images of testing material for the implantation periods (3,7,14, and 28) days are illustrate in figure (1).

Groups Comparison Statistically
The mean values and the frequency of the inflammatory cells scores and the proportion of fibrous capsule thickness for the (-ve) control group (empty tube), (+ve) control group (BioRoot sealer), and experimental sealer group at different observation times were represented in table (1).Kruskal-Wallis test was done at 0.05 significant to compare the effect of each material on the severity of tissues reaction.
Kruskal Wallis test revealed statistically significant differences among the material's effects on the inflammatory tissue reaction at (3, 7, and 14) day as shown in table (2).
Mann-Whitney test was used at 0.05 significant levels to see which pairs of groups differ significantly as shown in table (3).

DISCUSSION
When sealer extruded through the apical foramen and even when kept within the canal space, an inflammatory response of varying intensity usually develops in the area where the sealers contact the vital apical and periradicular tissues (16,17) .
After three day observation, moderate to severe infiltration of inflammatory cells (neutrophil infiltration) was observed in the (-ve) control and BioRoot sealer groups.These tissues response may be due to the trauma of the surgical procedure for tube implantation as reported in many studies (1,8) .However, the experimental sealer group showed moderate to mild infiltration of inflammatory cells; this less tissues reaction may be associated with the experimental sealer's composition and properties.
Periapical tissue reaction after root canal treatment may be influenced by various factors depending on the chemical nature of the endodontic sealer.Studies shown that most of bioceramic sealers have been found to be biocompatible and this was attributed to their ability to form Ca(OH)2, and Ca3(PO4)2 byproducts and this can promote the biomineralization and osseo-conductivity event when the sealer extruded through the apical foramen during root canal filling (2,18,19) .
In addition, alkaline pH could neutralize the lactic acid from osteoclasts and prevent dissolution of mineralized components; this will in turn stimulate the deposition of hard tissue and accelerate the healing process (5,12) .
From other side, studies showed that the use of scaffold containing extracellular matrix protein that containing acidic amino acid like (Aspartic and Glutamic) will enhance the cell adhesion, proliferation, and differentiation (20,21) .
Studies reported that the nanosized sealer's particles may promote the adhesion, differentiation, and proliferation of the cells leading to a faster repair and healing (9,10) .
After seven days observation the (ve) control and experimental sealer groups exhibited a reduction in inflammatory cell infiltration, while the BioRoot sealer group exhibited moderate inflammatory infiltration and this may be due to highly solubility for BioRoot sealer (8,22) .
After fourteen-day observation, the The rabbits were housed in private veterinarian clinic throughout the study time and under supervision of veterinary physician for preserving their health and habitual situation.Each rabbit was generally anesthetized using intramuscular administration of a rodent anesthesia.The dorsal skin of the rabbits was shaved and disinfected.Subcutaneous trichotomy 10 mm lengths were made with a sterile blade No. 10 on the back of rabbits along the spine in a head-tail orientation.Two incisions were made on the left half and one on the right half 2 cm from the spine and 4 cm apart from each other.Each animal received three polyethylene tubes (1.5 mm inner diameter, 10 mm length); one tube filled with BioRoot sealer (+ve control) was implanted on the left side near the head, and one tube filled with the experimental sealer was implanted on the right side near the head.The tube filled with standard sealer mass and inserted immediately to each corresponding incised pocket.The third incision received an empty tube as a (-ve) control implanted on the left side near the tail.After that the margins of the wounds were closed with 3/0 black silk suture, and disinfected with Oxytetracyclin solution.At the end of each implanted periods five animals were sacrificed by anesthetic overdose.The tubes were removed together with the surrounding tissues as a block section (20 x 20 mm), and fixed in thickness longitudinal serial sections using microtome, and then the resultant slides were stained with hematoxylin and eosin.Histological analysis for all slides was performed by experienced oral pathologist blinded to materials type and implantation intervals using Olympus light microscopy at 40X magnification.The intensity and the degree of inflammatory reaction were evaluated at the connective tissue adjacent to the open end of tube.The inflammatory events were scored based on the following FDI criteria (15) : Grade 0: No inflammatory cells or presence of less than 5 cells.Grade 1: Mild inflammation (5-25) inflammatory cells.Grade 2: Moderate inflammation (25-125) inflammatory cells.Grade 3for fibrous capsule; thickness was evaluated using micrometer lens at 40X and considered as thin when it was < 150 μm and thick when it was > 150 μm.Kruskal-Wallis test was used to evaluate the scores of inflammatory tissues reaction and Mann-Whitney test were done at 0.05 significant levels to comparison the difference in tissues response among groups at different implantation period.
The groups showed different tissues inflammatory response.However, all the groups showed decreased in the inflammatory tissues response over the times after implantation until no tissue inflammatory reactions remained at 28 day as represent in figure(2).

Figure ( 2 )
Figure (2): Histogram Representing the Evanescence of Inflammatory Tissues Response over Observation times.

(
-ve) control and BioRoot sealer groups exhibited more tendencies to mild inflammatory tissues reaction, while the experimental sealer group exhibited mild to absence inflammatory infiltration.However, all groups exhibited large amounts of collagen fibers and new blood vessels indicating normal tissues formation, although the experimental sealer group represented the larger amount of fibroblast cell and the more organized collagen fiber.The intensity of the reaction was diminished by the day 14, and this reduction continued progressively through

Table ( 1
): Mean and Standard deviation of the Inflammatory Tissues Response for Testing Groups at different Observation Periods * Statistically Significant Differences; NS Not Significant SD: Standard Deviation

Table ( 2
): Kruskal-Wallis Test for Comparison the Mean for Severity of Inflammation of Different Materials after Implantation Periods