Reaction of Rat Subcutaneous Connective Tissue to a Mineral Trioxide Aggregate–based and a Zinc Oxide and Eugenol Sealer Osvaldo Zmener, DDS, Dr Odont,* Ricardo Martinez Lalis, DDS,* Cornelis H. Pameijer, DMD, MScD, DSc, PhD,‡ Carolina Chaves, DDS,* Gabriel Kokubu, DDS,† and Daniel Grana, DVM† Abstract Introduction: The purpose of this study was to evaluate the subcutaneous connective tissue reaction in rats to a mineral trioxide aggregate (MTA)-based endodontic sealer Fillapex (Angelus, Londrina, PR, Brazil) and compare it with Grossman sealer (Farmadental, Buenos Aires, Argentina). Methods: Sterile medical-grade silicone tubes containing the test materials were implanted in 24 Wistar rats. After 10, 30, and 90 days, the animals (n = 8 per period) were euthanized, and the implants along with their surrounding tissues were dissected, fixed, and processed for histologic evaluation. A 4category evaluation system was used to evaluate the microscopic observations. The tissue response on the lateral walls of the silicone tubes was used as the negative control. The data were analyzed for statistical significance using the Wilcoxon signed rank, Kruskal-Wallis, and Dunn tests. Results: Fillapex showed a severe tissue reaction for all 3 observation periods. Grossman sealer showed similar features after 10 and 30 days, but the reaction decreased slightly after 90 days. In contrast, the negative controls did not show adverse reactions in any sample of the 3 time periods. After 10 and 30 days, no statistically significant differences were found between Fillapex and Grossman sealer (P > .05); however, the difference was significant after 90 days (P < .05). For all experimental periods, there were statistically significant differences between both Fillapex and Grossman sealer and the negative controls (P < .05). Conclusions: It was concluded that both MTA-Fillapex and Grossman sealer remained toxic to subcutaneous tissues in rats after 90 days. (J Endod 2012;38:1233–1238)
Key Words Biocompatibility, endodontics, Grossman sealer, mineral trioxide aggregate, subcutaneous connective tissue
fter proper debridement and disinfection, the root canal space must be filled with biocompatible materials (1, 2). During the last decade, mineral trioxide aggregate (MTA), a calcium silicate–based material, has gained popularity in endodontics as a root-end filling and pulp-capping material (3–11), for the repair of root canal perforations (12), and for apexification (13). Previous reports have shown that MTA is biocompatible (14–18), noncytotoxic (19–21), nonmutagenic, and neither genotoxic nor carcinogenic (22–24). In addition, MTA has shown antibacterial (25) and excellent sealing properties (18). Based on these favorable characteristics, MTA-based endodontic sealers suitable for root canal obturation have been developed recently (26) for the purpose of improving the drawbacks of the conventional MTA formulation such as the long setting time and the difficulty when handling it in the root canal. Among these new formulations, the MTA-based sealer Fillapex (FLPX; Angelus, Londrina, PR, Brazil) has been introduced recently. The manufacturer lists the formulation as follows: MTA, salicylate resin, natural and diluting resins, nanoparticulated resin, bismuth trioxide, nanoparticulated silica, and pigments (27). The manufacturer further claims low solubility, radiopacity, easy handling, and extended working time. To the best of our knowledge, little information is available on the effect of FLPX sealer when in contact with living tissues. Therefore, the purpose of this study was to evaluate the biocompatibility of FLPX when implanted in subcutaneous connective tissues in rats and compare it with Grossman sealer (Farmadental, Buenos Aires, Argentina). The latter is a well-established zinc oxide and eugenol material.
Materials and Methods The protocol for this study was reviewed and approved by the Research Ethics Committee of the Argentine Dental Association, Buenos Aires, Argentina. Forty-eight autoclaved silicone tubes (Raholin SRL, Buenos Aires, Argentina) measuring 10 mm long with an internal diameter of 1 mm were allocated to 2 groups of 24 tubes each (n = 24). In 1 group, the tubes were filled flush with freshly prepared FLPX, whereas the other group contained Grossman sealer, which served as the positive control. The lateral walls of the silicone tubes were used as the negative controls (NCs). Care was taken to keep the test materials within the tubes and to prevent contamination of the lateral wall. The sealers were prepared under aseptic conditions and mixed according to the manufacturers’ instructions.
From the *Postgraduate Program for Specialized Endodontics and †Department of Oral Pathology, Faculty of Medical Sciences, School of Dentistry, University of El Salvador, Buenos Aires, Argentina; and ‡University of Connecticut School of Dentistry, Farmington, Connecticut. Supported in part by a grant from the Argentine Dental Association (AO367–2011). Address requests for reprints to Dr Cornelis H. Pameijer, University of Connecticut School of Dental Medicine, 10 Highwood, Simsbury, CT 06070. E-mail address: [email protected]
0099-2399/$ - see front matter Copyright ª 2012 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2012.05.010
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Basic Research—Technology TABLE 1. Distribution of the Implants and the Severity of Tissue Reaction to Materials FLPX
Days n NO MI MO SE NO MI MO SE NO MI MO SE 10 30 90
8 8 8
– – –
– – –
– – –
8 8 8
– – –
– – –
– – 7
8 8 1
8 8 8
– – –
– – –
– – –
GS, Grossman sealer; MI, mild reaction; MO, moderate reaction; NO, no reaction; SE, severe reaction.
After complete filling, the tubes were immediately implanted into the subcutaneous connective tissue of 24 white male Wistar rats that weighed approximately 200 g each. The husbandry and management of the animals met the requirements of the ISO 10993-1 (1992) and ISO 10993-2 (1992) standards (28, 29) as well as the International Regulatory Requirements for the care and use of laboratory animals (30). Every effort was made to minimize animal discomfort and limit the total number of animals used. All operative procedures were performed under strict aseptic conditions. The surgical procedures were as follows. General anesthesia was administered through an intraperitoneal injection of ketamine chloride and acepromazine (14 mg/10 mg/kg body weight). The dorsal skin was shaved and disinfected with 5% iodine in alcohol. An 18-mm-long incision was made through the skin, and 2 separate pockets were prepared by blunt dissection. One sample of each material was placed into the pockets of each rat to a depth of 20 mm from the line of the incision and was sufficiently separated from the other sample to avoid interference of the tissue response. Subsequently, the wounds were closed with silk sutures, and the animals were maintained in cages
on a regular diet and water ad libitum. The animals were euthanized in groups of 8 after 10, 30, and 90 days with an anesthetic overdose. The implants with surrounding tissues were carefully dissected and fixed in 10% neutral buffered formalin (pH = 7.4). The solution was replenished after 24 hours, and the samples were fixed for 7 days. After fixation, the tissues were processed for paraffin embedding. The paraffin blocks were oriented parallel to the long axis of the tubes, and longitudinal serial sections of approximately 7 mm thick were obtained from the central areas of the implants. The histologic sections were stained with hematoxylin and eosin. To evaluate the tissue response in the areas of tissue/material contact, 3 sections belonging to the central areas of each specimen were analyzed and photographed at different magnifications with a light microscope equipped with a digital Canon Powershot A510 camera (Canon, Tokyo, Japan). These sections were analyzed blind by 2 trained evaluators who independently scored the thickness of a fibrous capsule, the vascular changes, and the various types of inflammatory cells. The tissue reactions were scored as follows: (1) no reaction, fibrous capsule formation and the absence of inflammatory cells; (2) mild reaction, the presence of a fibrous capsule formation with few inflammatory cells; (3) moderate reaction, fibrous capsule formation with the presence of polymorphonuclear leukocytes, lymphocytes, plasmocytes, and macrophages; and (4) severe reaction, the absence of a fibrous capsule with the presence of large accumulations of polymorphonuclear leukocytes, lymphocytes, plasmocytes, macrophages, foreign-body giant cells, and congested capillaries. Before the analysis, both evaluators were calibrated and had to analyze a set of 70 similar but unrelated slides that had inflammatory reactions to different endodontic sealers. If there was a disagreement between the evaluators, the sample under discussion was analyzed jointly until
Figure 1. (A–D) Representative specimens of FLPX and Grossman sealer at the 10-day observation. (A) FLPX: an overview of the area of tissue/material contact showing a slight invagination of granulomatous tissue (hematoxylin and eosin stain; original magnification, 100). (B) A higher magnification of the outlined area in A. There is a thin fibrous connective band containing inflammatory cells (white arrow) in direct contact with numerous particles of the sealer (black arrow). Below the fibrous connective band, the tissue reaction is still severe (hematoxylin and eosin stain; original magnification, 1,000). (C) Grossman sealer: an overview of the area of tissue/material contact showing a severe granulomatous tissue reaction invaginated within the lumen of the tube (hematoxylin and eosin stain; original magnification, 100). (D) A higher magnification of outlined area in C. Note the severe granulomatous tissue reaction containing polymorphonuclear leucocytes, lymphocytes, plasmocytes, fibroblasts, macrophages, and newly formed capillaries. Note also the presence of a multinucleated giant cell (arrow) (hematoxylin and eosin stain; original magnification, 1,000).
Zmener et al.
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Basic Research—Technology a consensus was reached. Data were analyzed by the Wilcoxon signed rank test to determine if there was a statistically significant difference between FLPX, Grossman sealer, and NCs at each observation period. The total effect of time and material upon the tissue reaction was calculated by the Kruskal-Wallis and the Dunn test. The significance level was set at P < .05. At the end of the experiment, the sealers were considered biologically acceptable when the tissue reaction was recorded as none to mild.
Results Macroscopic examination of the implantation sites revealed that wound healing was satisfactory for all time periods. The number and distribution of the implants as well as the severity of tissue reaction are presented in Table 1.
NCs After 10 days, the lateral walls of the test tubes were surrounded by thin fibrous connective tissues, the thickness of which progressively increased over 30 and 90 days without inflammatory cells. It could be distinguished easily from the tissue reaction at the sites where the tissues were in direct contact with FLPX and Grossman sealer.
Experimental FLPX and Positive Grossman Sealer Control Samples After 10 days, the inflammatory tissue reaction to FLPX and Grossman sealer were severe (Fig. 1A–D). When in direct contact with FLPX,
a slight tissue invagination into the lumen of the tubes was observed with many newly formed vessels and randomly dispersed dark particles, which appeared to have been released from the material. The particles were surrounded by numerous polymorphonuclear leukocytes, lymphocytes, some plasmocytes, and macrophages. This dense granulomatous tissue had a high concentration of polymorphonuclear neutrophils, lymphocytes, plasmocytes, macrophages, and an occasional multinucleated giant cell. After 30 days, the intensity of the inflammatory reaction of tissues in contact with FLPX and Grossman sealer was also severe (Fig. 2A– D). Both materials showed persistent but localized granulomatous tissues invaginated into the lumen of the tubes. This reaction was distinctly separated from the rest of the tissues by fibrous tissues that were free of inflammatory cells. The samples of FLPX showed a fibrogranulomatous tissue containing many dispersed or clusters of material particles surrounded by macrophages and multinucleated giant cells together with lymphocytes, fibroblasts, and a few polymorphonuclear leukocytes. In direct contact with Grossman sealer, dark masses of material were seen within the granulomatous tissue, which were surrounded by numerous inflammatory cells, mostly lymphocytes plasmocytes, fibroblasts, and macrophages. After 90 days, a severe granulomatous tissue invagination still persisted in all FLPX samples. However, 7 Grossman sealer samples had a moderate inflammatory score (Fig. 3A–D), whereas only 1 was severe. In contact with FLPX, lymphocytes, plasmocytes, and numerous macrophages and multinucleated giant cells containing material particles within their cytoplasm were observed. Somewhat similar features
Figure 2. (A–D) Representative specimens of FLPX and Grossman sealer at the 30-day observation. (A) FLPX: an overview of the area of tissue/material contact. There is a dense fibrogranulomatous tissue invagination within the lumen of the tube showing a clear separation of an area of fat cells and the rest of the healthy tissues (arrow) (hematoxylin and eosin stain; original magnification, 100). (B) A higher magnification of outlined area in A. There is a dense fibrogranulomatous tissue containing many randomly distributed particles (white arrows) or phagocytized by macrophages (black arrow) (hematoxylin and eosin stain; original magnification, 1,000). (C) Grossman sealer: an overview of the area of tissue/material contact showing a dense granulomatous tissue invagination containing gross masses of material within the lumen of the tube. Note clear separation from the surrounding healthy tissues (hematoxylin and eosin stain; original magnification, 100). (D) A higher magnification of outlined area in C. Note the presence of many dense material particles within a severe granulomatous tissue reaction (hematoxylin and eosin stain; original magnification, 1,000). JOE — Volume 38, Number 9, September 2012
Tissue Reaction to an MTA-based Sealer
Figure 3. (A–D) Representative specimens of FLPX and Grossman sealer after 90 days. (A) FLPX: an overview of the area of tissue/material contact showing a severe granulomatous tissue invagination within the lumen of the tube (hematoxylin and eosin stain; original magnification, 40). (B) A higher magnification of the area of arrow in A. In the contact area, a dense concentration of inflammatory cells mainly composed by macrophages (white arrows) and multinucleated giant cells (black arrows) can be seen containing material particles in the cytoplasm (hematoxylin and eosin stain; original magnification, 1,500). (C) Grossman sealer: an overview of the area of tissue/material contact showing a dense fibrous connective capsule and below it a high concentration of inflammatory cells (hematoxylin and eosin stain; original magnification, 100). (D) A higher magnification of outlined area in C. Note the thick fibrous connective tissue capsule containing a few inflammatory cells (arrow) and below it a considerable concentration of inflammatory cells mainly composed of macrophages with phagocytized material particles into their cytoplasm (hematoxylin and eosin stain; original magnification, 1,000).
were observed for 2 of the Grossman sealer samples, but 6 samples differed from FLPX in that a dense fibrous connective tissue layer (50–80 mm thick) that contained a few inflammatory cells was observed. Beyond this connective tissue layer, numerous inflammatory cells, mainly composed of lymphocytes and macrophages that contained phagocytized particles, were seen randomly distributed into the tissues. The Wilcoxon signed rank test showed no statistically significant differences between FLPX and Grossman sealer after 10 and 30 days (P > .05). However, after 90 days, a difference was shown between FLPX and Grossman sealer (P < .05), whereas both materials significantly differed from the control in all observation periods. The total effect of time for FLPX and Grossman sealer revealed that the results obtained after 10 and 30 days differed significantly from the 90-day observation period (P < .05). Conversely, no significant differences were observed for the NCs of the 3 time intervals (P > .05).
Discussion Implantation of endodontic filling materials into the subcutaneous connective tissue in rats is considered a valid secondary screening test for biocompatibility (31, 32). The implantation periods in this study were within the short- and long-term time intervals of the recommended standards practices for biological evaluation of dental materials (33). Grossman sealer was used as the positive control because the toxicity of zinc oxide and eugenol materials has been determined previously by ex vivo (34, 35) and in vivo experiments (36, 37). The lateral walls of the silicone tubes were used as the NC because the material 1236
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by itself has been proven to be biocompatible (38–40), a finding that was confirmed in the current study. In a study by Scarparo et al (41), Endo-CPM Sealer (EGEO SRL, Buenos Aires, Argentina), an MTA-based sealer; gray MTA (Pro Root; Dentsply, Tulsa, OK); and an epoxy resin-based sealer (AH Plus, Dentsply) were implanted in the subcutaneous connective tissue of rats. After 60 days, the authors reported scar and dense fibrous tissues in contact with MTA and the MTA-based sealer without inflammatory cells (41), whereas an intense chronic inflammation was observed in contact with the epoxy resin–based sealer. These results are in support of previous findings by Martinez Lalis et al (42). In the present study, the severity of the tissue reactions persisted even after 90 days. These differences may be because of the composition of the tested materials. Endo-CPM sealer (a powder/liquid formulation) is similar to MTA, and the liquid component is a saline solution with calcium chloride. FLPX is marketed as a paste/paste in a double-barrel automix system and contains MTA, resins, and other components. Bramante et al (43) showed that many MTA formulations contain high levels of arsenic, a heavy metalloid with a known toxicity to cells (44). However, they found that white MTAAngelus (Angelus, Londrina, PR, Brazil) contained arsenic levels within the ISO 9917-1–recommended limits (45). According to the manufacturer, the amount of MTA in FLPX was reduced to 30%. Therefore, it is unclear whether the release of arsenic from the reduced amount of MTA in the sealer caused the severe reaction or whether other components are responsible. Further investigations are warranted. Another component of potential concern is the presence of resins in FLPX. As per protocol, the material was implanted in its freshly mixed state. It is possible that unpolymerized resins after degradation by enzymatic JOE — Volume 38, Number 9, September 2012
Basic Research—Technology action (46, 47) leading to toxic byproducts could have leached into the surroundings, thus causing the severe inflammatory reactions at the 10- and 30-day evaluation period. Of concern here, however, is that the severity of the inflammatory reaction persisted to the end of the experiment (90 days) and that numerous particles of the material were still present in the tissues surrounding the implants or within the cytoplasm of macrophages and multinucleated foreign-body giant cells. In a preliminary pilot study (Drs Zmener and Martinez Lalis, unpublished data, 2012), the solubility of FLPX was tested according to the standards of the American Dental Association specification no. 57 for endodontic filling materials (48), which states that the solubility of a set material shall not exceed 3% by mass. FLPX showed a solubility of 3.95%, exceeding the ADA recommendations. Therefore, we can speculate that after the initial rapid loss further leaching will occur because of its high solubility. The tissue reaction to Grossman sealer was rated as severe after 10 and 30 days, which, as has been shown (49, 50), is caused by traces of unreacted eugenol exerting a toxic reaction to cells (34, 35). After 90 days, there is a reduced availability of unreacted eugenol allowing for the formation of fibrous connective tissues and an inflammatory score from severe to moderate. The findings of Grossman sealer reported here are in agreement with other reports on zinc oxide and eugenol sealers (36, 37, 51, 52). Despite a reduction of severity, GC remained toxic.
Conclusions Within the limits of this study, it was concluded that FLPX and GC when implanted in subcutaneous tissues in rats remained toxic even after 90 days.
Acknowledgments The authors thank Professor Renato Miotto Palo for his collaboration with this experiment. The authors deny any conflicts of interest related to this study.
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