Histopathological evaluation of bone tissue response to endodontic sealers and gutta-percha using an intraosseous implantation model in Wistar rats

Abstract

Aim:

To comparatively evaluate the biological response of bone tissue to different endodontic sealers and gutta-percha (GP) using an intraosseous implantation model in Wistar rats.

Materials and Methods:

Bone tissue responses to six endodontic materials, resin-based sealer (Seal Pex), calcium hydroxide-based sealer (Sealapex), silicone-based sealer (GuttaFlow 2), calcium silicate-based sealer (Ceraseal), MTA-based sealer (MTA Fillapex), and GP, were evaluated in 21 Wistar rats. An empty cavity served as the control. Standardized bone cavities were prepared in the femur and filled with the test materials. Histological evaluation was performed at 7, 30, and 90 days (n = 3) by assessing inflammatory cell infiltration, collagen fiber formation, hard tissue barrier formation, abscess formation, and material polarization. Data were analyzed using Fisher’s exact test (P < 0.05).

Results:

All materials induced an acute inflammatory response at 7 days. Resin-based sealer and MTA Fillapex showed significantly higher inflammatory cell infiltration, giant cell presence, and abscess formation. At 30 and 90 days, calcium hydroxide-based, calcium silicate-based, and silicone-based sealers, along with GP, demonstrated reduced inflammation, improved collagen organization, and advanced hard tissue barrier formation. In contrast, resin-based sealer and MTA Fillapex exhibited persistent inflammation and delayed bone healing.

Conclusion:

Bone tissue response to endodontic obturation materials was material- and time-dependent. Although all tested materials are routinely used in clinical practice, calcium hydroxide-based, calcium silicate-based, and silicone-based sealers, along with GP, exhibited a more favorable biological response over time. The comparatively prolonged inflammation observed with resin-based sealer and MTA Fillapex underscores the need for careful material selection and controlled obturation, particularly in cases with periapical involvement.

INTRODUCTION

Successful endodontic treatment depends on thorough cleaning and shaping of the root canal system, followed by complete obturation of the prepared canal space using an inert filling material. Among the various obturation techniques, the combination of gutta-percha (GP) with an endodontic sealer is widely recognized as a reliable and effective approach.[1] The sealer plays a vital role by filling voids, anatomical irregularities, and accessory canals that GP alone may not adequately seal, thereby enhancing the three-dimensional seal and reducing the risk of microleakage and microbial reinfection.[2]

Due to the close relationship between the dental pulp and periodontal tissue, clinicians must carefully consider the anatomical pathways that connect these tissues. As the tooth matures and root development is completed, communication pathways such as dentinal tubules, lateral and accessory canals, and the apical foramen are established. Consequently, obturating materials come into direct contact with periodontal tissue through these pathways.[3]

Obturating materials are intended to stay confined within the root canal during treatment, but extrusion into surrounding tissues is a common clinical occurrence.[4,5] When in contact with periapical tissues, GP and sealers can act as foreign bodies, triggering connective tissue responses.[6] The extent and type of tissue reaction depend on factors such as the material’s type and quantity, the location of extrusion, and the tissue’s condition.[7] Studies have shown that materials with lower cytotoxicity and anti-inflammatory properties reduce adverse tissue reactions, promoting better healing outcomes. Furthermore, the chemical composition of the sealer significantly influences its biocompatibility and interaction with periapical tissues.

Sealers are categorized based on their chemical composition, including zinc oxide eugenol, calcium hydroxide, MTA, silicone, resin, and calcium silicate-based sealers, each offering distinct properties that influence their clinical performance.[8] While zinc oxide eugenol sealers are valued for their antimicrobial activity, their tendency to dissolve over time is a limitation. Calcium hydroxide sealers are known for their ability to stimulate hard tissue formation, but may lack long-term stability. MTA-based sealers demonstrate excellent sealing and tissue repair properties, although their handling and cost can pose challenges. Silicone-based sealers are dimensionally stable and user-friendly, but offer limited antimicrobial efficacy. Resin-based sealers, with their superior adhesive properties, ensure a tight seal but may be cytotoxic and difficult to remove during retreatment. Calcium silicate-based sealers, characterized by their bioactivity and ability to integrate with surrounding tissues, are increasingly explored for their potential in enhancing biological responses. The variation in these sealers’ properties underscores the importance of evaluating their interactions with tissues, such as connective tissue and bone, to guide their selection in clinical practice.

Numerous studies employing various experimental methods have evaluated the tissue response to endodontic materials. Subcutaneous implantation of these materials, with or without carriers, helps simulate the stabilization process and interaction with connective tissue in situ and is useful for understanding the initial inflammatory and immune responses; however, this model does not accurately represent the biological response of alveolar bone tissue.[9,10,11,12] In contrast, intraosseous implantation of endodontic materials provides a more suitable model for evaluating material biocompatibility, as it more closely simulates clinical conditions and allows assessment of bone healing and regeneration over time.

Despite the availability of various endodontic sealers, differences in their chemical composition may lead to variable biological responses when these materials come into contact with bone tissue. While several studies have focused on tissue compatibility using subcutaneous models, evidence regarding bone tissue response remains limited. Therefore, the present in vivo study aimed to comparatively evaluate the bone tissue response to different endodontic sealers and GP using an intraosseous implantation model at 7, 30, and 90 days.

MATERIALS AND METHODS

The study was approved by the Institutional Animal Ethics Committee of CLART under WBLDCL. Bone tissue responses to six endodontic materials were evaluated in 21 Wistar rats, with three animals randomly assigned to each of the following groups: resin-based sealer (Seal Pex), calcium hydroxide-based sealer (Sealapex), silicone-based sealer (GuttaFlow 2), calcium silicate-based sealer (Ceraseal), MTA-based sealer (MTA Fillapex), GP, and a negative control group with an empty cavity. Evaluations were performed after 7, 30, and 90 days (n = 3 per period). Anesthesia was achieved using ketamine (0.008 mL/100 g) and 2% xylazine hydrochloride (0.004 mL/100 g). The right femur served as the surgical site. Following trichotomy, the area was disinfected with an alcohol–iodine solution, and a 5-cm skin incision was made. Soft tissues were separated in layers, and the periosteum was incised. Three cortical cavities, each 6 mm in diameter, were prepared on the femoral surface. The sealers were mixed according to the manufacturers’ directions and transferred into insulin syringes, and 0.2 mL of the respective material was placed into each cavity. The incision was sutured in layers, and postoperative analgesia was provided with an intramuscular injection of 50 mg/kg opioid. At the end of each experimental period, the animals were euthanized using CO₂ inhalation. The operated limb was disarticulated, and the femur was isolated. Using a slow-rotation diamond disc, the bone was transversely sectioned to obtain the region containing the cavities. Each specimen was fixed in 10% neutral-buffered formalin for 48 h and subsequently decalcified in 10% nitric acid. The samples were embedded in paraffin, and transverse sections of 5 µm thickness were prepared. The slides were stained with hematoxylin and eosin and examined under a light microscope at 40×, 100×, 200×, and 400× magnifications. The repair process was evaluated based on histologic findings, including the characteristics of inflammatory cell infiltration, fibrous tissue formation, and the presence of a hard tissue barrier beneath the cavity opening.

Cellular reactions and collagen fiber organization were assessed qualitatively based on previously established histologic criteria by Tavares et al.[13] The inflammatory cellular component was identified by the presence of neutrophils, lymphocytes, eosinophils, macrophages, and multinucleated giant cells. The intensity of cellular response was graded as follows: (0) absence, when inflammatory cells were not observed or were confined to blood vessels; (1) mild, when cells were present in small numbers or scattered clusters; (2) moderate, when inflammatory cells were evident but did not predominate within the microscopic field; and (3) intense, when dense inflammatory infiltrates were observed in close proximity to the implanted material.

Collagen fiber condensation was evaluated according to the thickness and organization of the fibrous tissue and categorized as (0) absence of collagen fibers, (1) presence of a thin collagen layer, or (2) presence of a thick collagen layer. Abscess formation was assessed and classified as (1) absent, (2) present in direct contact with the surgical cavity containing the material, or (3) present at sites distant from the surgical cavity.

Hard tissue barrier formation was evaluated using a modified scoring system based on Assmann et al.[14] The response was classified as (1) absence, when no mineralized tissue deposition was observed within the cavity region; (2) immature bone formation, characterized by early mineralized tissue indicating the initiation of linear closure of the experimental defect; (3) partial formation, when the cavity was incompletely closed by newly formed hard tissue; and (4) complete formation, when the defect was entirely closed by mineralized tissue. In addition, material polarization was recorded and categorized as either positive or negative.

RESULTS

Bone tissue response differed significantly among materials and time points (P < 0.05) [Tables 1 and 2]. At 7 days, all materials elicited an acute inflammatory response. Resin-based sealer and MTA Fillapex showed higher inflammatory cell infiltration, frequent giant cells, and increased abscess formation, whereas calcium hydroxide, calcium silicate, and silicone-based sealers, along with GP, demonstrated milder inflammation with minimal hard tissue formation. By 30 days, inflammation decreased in most groups. Calcium hydroxide, Calcium silicate, and silicone-based sealers, as well as GP, exhibited improved collagen organization and partial hard tissue barrier formation, while resin-based sealer and MTA Fillapex showed persistent inflammation and delayed bone repair [Figure 1].

Table 1.

Absolute frequencies of inflammatory cellular events in bone tissue response to different endodontic materials at 7, 30, and 90 days

Time	Materials	Neutrophils	Lymphocytes	Eosinophils	Macrophages	Giant cells
7 days	Resin based	4 (2) 3 (1)	2 (3)	3 (2) 2 (1)	3 (2) 2 (1)	2 (3)
	Calcium hydroxide based	2 (3)	1 (3)	3 (2) 2 (1)	2 (3)	2 (3)
	Calcium silicate based	2 (3)	2 (3)	3 (3)	2 (3)	2 (3)
	MTA based	3 (2) 2 (1)	2 (3)	3 (2) 2 (1)	3 (3)	4 (2) 2 (1)
	Silicone based	3 (3)	2 (3)	3 (3)	3 (2) 2 (1)	3 (3)
	Gutta-percha	4 (2) 3 (1)	2 (3)	2 (3)	2 (3)	3 (3)
	Control	1 (3)	1 (3)	1 (3)	1 (3)	1 (3)
	P	<0.001	<0.001	0.003	<0.001	<0.001
30 days	Resin based	3 (3)	2 (3)	2 (3)	2 (3)	2 (3)
	Calcium hydroxide based	2 (3)	2 (3)	2 (3)	1 (3)	1 (3)
	Calcium silicate based	1 (3)	2 (3)	1 (3)	2 (3)	2 (3)
	MTA based	4 (2) 3 (1)	3 (3)	2 (3)	3 (3)	2 (3)
	Silicone based	1 (3)	1 (3)	2 (3)	1 (3)	1 (3)
	Gutta-percha	2 (3)	2 (3)	2 (3)	2 (3)	1 (3)
	Control	2 (3)	1 (3)	2 (3)	1 (3)	2 (3)
	P	<0.001	<0.001	0.005	<0.001	<0.001
90 days	Resin based	3 (2) 2 (1)	2 (3)	2 (3)	2 (3)	2 (3)
	Calcium hydroxide based	1 (3)	1 (3)	2 (3)	2 (2) 1 (1)	1 (3)
	Calcium silicate based	2 (3)	3 (3)	1 (3)	2 (3)	2 (3)
	MTA based	4 (2) 3 (1)	2 (3)	3 (3)	3 (3)	3 (3)
	Silicone based	1 (3)	1 (3)	1 (3)	1 (3)	1 (3)
	Gutta-percha	1 (3)	1 (3)	1 (3)	1 (3)	1 (3)
	Control	1 (3)	1 (3)	1 (3)	1 (3)	1 (3)
	P	<0.001	<0.001	<0.001	<0.001	<0.001

Values are expressed as histologic grading scores. Numbers in parentheses indicate the number of animals (n) exhibiting the respective score at each time interval

Table 2.

Distribution of histologic bone tissue response parameters and material polarization in response to different endodontic materials at 7, 30, and 90 days

Time	Materials	Collagen fibers	Hard tissue barrier	Abscess formation	Material polarization
7 days	Resin based	2 (3)	1 (3)	2 (3)	Positive
	Calcium hydroxide based	1 (3)	1 (3)	1 (3)	Positive
	Calcium silicate-based	3 (3)	2 (3)	1 (3)	Positive
	MTA based	2 (3)	2 (2) 1 (1)	2 (3)	Negative
	Silicone based	1 (3)	2 (3)	1 (3)	Positive
	Gutta-percha	3 (3)	3 (3)	2 (3)	Negative
	Control	2 (3)	2 (3)	1 (3)
	P	<0.001	<0.001	<0.001
30 days	Resin based	2 (3)	2 (3)	2 (3)	Positive
	Calcium hydroxide based	3 (3)	3 (3)	1 (3)	Positive
	Calcium silicate based	1 (3)	2 (3)	1 (3)	Positive
	MTA based	2 (3)	2 (3)	2 (3)	Negative
	Silicone based	3 (3)	3 (3)	1 (3)	Positive
	Gutta-percha	3 (2) 2 (1)	3 (3)	1 (3)	Negative
	Control	1 (3)	4 (2) 3 (1)	1 (3)
	P	<0.001	<0.001	<0.001
90 days	Resin based	2 (3)	3 (3)	2 (3)	Positive
	Calcium hydroxide based	1 (3)	4 (3)	1 (3)	Positive
	Calcium silicate-based	1 (3)	2 (3)	2 (3)	Positive
	MTA based	2 (3)	2 (3)	2 (3)	Negative
	Silicone based	2 (3)	3 (3)	1 (3)	Positive
	Gutta-percha	1 (3)	4 (3)	1 (3)	Negative
	Control	1 (3)	4 (3)	2 (3)
	P	<0.001	<0.001	<0.001

Values represent histologic grading scores, with numbers in parentheses indicating the number of animals (n=3) showing each score. Material polarization is presented as positive or negative

Figure 1.

Representative hematoxylin and eosin-stained photomicrographs showing bone tissue response to different endodontic materials following intraosseous implantation. (A) 7 days and (B) 30 days illustrate tissue reactions to (a) silicone-based sealer, (b) MTA-based sealer, (c) gutta-percha, (d) Calcium silicate based sealer, (e) calcium hydroxide-based sealer, and (f) resin-based sealer, demonstrating time-dependent changes in inflammatory cell infiltration, collagen organization, and progression toward tissue repair

At 90 days, silicone-based sealer, calcium hydroxide-based sealer, GP, and control specimens demonstrated minimal inflammation and advanced or complete hard tissue barrier formation, whereas resin-based sealer and MTA Fillapex continued to show persistent inflammation and incomplete bone healing [Figure 2].

Figure 2.

Representative hematoxylin and eosin-stained photomicrographs showing bone tissue response following intraosseous implantation. (A) Bone tissue response at 90 days to different endodontic materials: (a) silicone-based sealer, (b) MTA-based sealer, (c) gutta-percha, (d) Calcium silicate-based sealer, (e) calcium hydroxide-based sealer, and (f) resin-based sealer. (B) Control group showing bone tissue response at (a) 7, (b) 30, and (c) 90 days, illustrating the normal healing pattern in empty cavities over time

Statistical analysis

Statistical analysis was performed using STATA version 17.0 (StataCorp, College Station, TX, USA). Fisher’s exact test was used to evaluate the association between the different endodontic materials and bone tissue responses. A P value of less than 0.05 was considered statistically significant.

DISCUSSION

One of the fundamental objectives of endodontic treatment is to preserve or promote the regeneration of the periapical and surrounding bone structures. Consequently, understanding the interaction between endodontic sealers and bone tissue is of significant clinical importance. In the present in vivo investigation, the bone tissue response to various endodontic materials was evaluated at 7, 30, and 90 days by assessing inflammatory cell infiltration, collagen fiber formation, hard tissue barrier development, abscess formation, and material polarization. The findings indicated that both the material type and the duration of implantation had a significant impact on tissue reactions, with statistically significant differences observed among groups for most parameters (P < 0.05).

At 7 days, all materials produced an acute inflammatory response, consistent with early healing. Resin-based sealer and MTA Fillapex demonstrated significantly higher inflammatory cell infiltration and giant cell presence (P < 0.001), indicating a stronger foreign body reaction. This response may be attributed to the release of residual resin monomers and other organic by-products during polymerization and degradation of resin-based sealers, which are known to exert cytotoxic effects on periapical cells and sustain inflammatory activity.[15] Similarly, MTA Fillapex, despite containing calcium silicate components, has been reported to induce higher cytotoxicity and inflammatory responses than other calcium silicate-based sealers, likely due to the presence of resinous salicylate constituents that may irritate surrounding tissues and delay the resolution of inflammation.[16]

In contrast, calcium hydroxide, calcium silicate, and silicone-based sealers exhibited milder inflammation, reflecting better early biocompatibility. Hard tissue barrier formation was minimal at this stage, while abscess formation was more common with resin-based sealer and MTA Fillapex. However, contrasting evidence has been reported, as Elias et al.[17] observed that the inflammatory tissue response induced by a bioceramic endodontic sealer was not milder than that produced by an epoxy resin-based sealer during the first 3 weeks, based on microscopic evaluation of the reactionary tissue.

By 30 days, inflammation significantly decreased in most groups, indicating progression toward tissue repair. Calcium hydroxide-, calcium silicate-, and silicone-based sealers, along with GP, showed reduced inflammatory scores, increased collagen fiber organization, and enhanced hard tissue barrier formation (P < 0.001). The favorable response observed with calcium hydroxide-based sealers may be attributed to hydroxyl ion release and the resultant alkaline pH, which, despite initial cytotoxicity, exerts antibacterial effects and promotes mineralization and subsequent hard tissue formation, thereby facilitating resolution of inflammation over time.[18] Silicone-based sealers demonstrated minimal tissue irritation, which may be explained by their chemical inertness, low solubility, and stable polymer network, limiting the release of irritating components and supporting favorable tissue healing. Similarly, calcium silicate-based sealers exhibited improved tissue compatibility, likely due to their bioactive hydration reaction and sustained release of calcium and hydroxyl ions, creating an alkaline environment conducive to cell viability, mineral deposition, and hard tissue formation adjacent to the material, thereby supporting osteogenic tissue repair over time.[19,20]

MTA Fillapex, however, continued to show moderate inflammation. Similar findings were reported by Tavares et al.[13] who demonstrated persistent tissue irritation associated with MTA Fillapex during the early and intermediate healing periods.

At 90 days, calcium hydroxide-based, silicone-based sealers, and GP exhibited minimal inflammation and advanced or complete hard tissue barrier formation, indicating superior long-term biocompatibility. These findings are supported by previous studies, where Veloso et al.[21] demonstrated favorable tissue response and progressive healing with calcium hydroxide-based sealers, and Santos et al.[22] reported limited inflammatory reactions and satisfactory biocompatibility with silicone-based bioceramic sealers in subcutaneous tissue. Bioceramic sealer showed stable tissue behavior with consistent mineralized tissue formation, consistent with the findings of de Miranda Candeiro et al.[23,24] who reported that calcium silicate-based sealers induced favorable tissue repair and minimal long-term inflammation compared with epoxy resin-based sealers.

CONCLUSION

Within the limitations of this in vivo intraosseous study, bone tissue response to endodontic materials was found to be both material- and time-dependent. All materials induced an initial inflammatory response at 7 days; however, calcium hydroxide, calcium silicate, and silicone-based sealers, along with GP, demonstrated a progressive reduction in inflammation, improved collagen organization, and enhanced hard tissue barrier formation at 30 and 90 days, indicating favorable biocompatibility. In contrast, resin-based sealer and MTA Fillapex showed persistent inflammation and delayed bone healing. These findings emphasize the importance of careful material selection in clinical situations where contact with bone tissue may occur.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.