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Year : 2014  |  Volume : 25  |  Issue : 1  |  Page : 22-27
Periapical tissue reaction to calcium phosphate root canal sealer in porcine model

1 Department of Conservative Dentistry and Endodontics, PSM College of Dental Science and Research, Akkikavu, Thrissur, Kerala, India
2 Department of Conservative Dentistry and Endodontics, Indira Gandhi Government Dental College, Jammu, Jammu and Kashmir, India
3 Department of Conservative Dentistry and Endodontics, Government Dental College, Trivandrum, Kerala, India
4 Principal and Professor, Dept. of Conservative Dentistry and Endodontics, PMS Dental College, Trivandrum, Kerala, India
5 Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Trivandrum, Kerala, India

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Date of Submission23-Jan-2013
Date of Decision18-Jul-2013
Date of Acceptance05-Dec-2013
Date of Web Publication21-Apr-2014


Background: Calcium phosphate cements (CPC) are a group of biomaterials possessing wide scope of use in various branches of medical science. These materials have been proposed to be highly biocompatible and osteoconductive. This study is based on a newly developed CPC formulation (Chitra-CPC) and is aimed at the evaluation of its biocompatibility through an Endodontic Usage Test in a porcine study model.
Objective: To evaluate the periapical tissue reaction to Chitra-CPC when used as a root canal sealer/filler material in comparison with a resin sealer, AH Plus (Dentsply).
Materials and Methods: The procedure was done on porcine animal model following the ISO 7405 criteria. The material was implanted intentionally into the periapical area of 36 teeth through a root canal procedure carried out in six animals which were divided equally among 1-month and 3-month time periods. Results were based on the histological evaluation of the autopsied specimens after the prescribed time periods.
Results: Mild to moderate periapical tissue reaction was found in Chitra-CPC samples belonging to the 1-month time period, whereas majority of the 3-month CPC samples showed an absence of inflammation. Samples of AH Plus in 1-month period showed severe to moderate inflammation, whereas 3-month AH Plus samples had a mild to moderate inflammation.
Conclusions: Chitra-CPC is a biocompatible material.

Keywords: Biocompatibility, calcium phosphate cement, Chitra-CPC, endodontic usage test, porcine model

How to cite this article:
Jacob GM, Kumar A, Varughese JM, Varghese N O, Harikrishna Varma P R, Komath M. Periapical tissue reaction to calcium phosphate root canal sealer in porcine model. Indian J Dent Res 2014;25:22-7

How to cite this URL:
Jacob GM, Kumar A, Varughese JM, Varghese N O, Harikrishna Varma P R, Komath M. Periapical tissue reaction to calcium phosphate root canal sealer in porcine model. Indian J Dent Res [serial online] 2014 [cited 2023 Sep 23];25:22-7. Available from:
The success and predictability of endodontic treatment lies in thorough shaping and cleaning of root canal system followed by proper three-dimensional obturation with a dimensionally stable, inert material so as to eliminate all possible portals of communication between the canal space and the surrounding periapical tissues. The accepted technique is to obturate the root canal space using a solid or semi-solid material along with a sealer to obtain a fluid tight seal, occupying the interstitial spaces, foraminae, as well as accessory and lateral canals. [1],[2],[3]

Over-extrusion of a root canal sealer may cause tissue degeneration and delay healing of the inflamed vital periapical tissues. [4] Even if sufficient care is taken to avoid extrusion, there is a sound chance for leachable substances or degradation products from the sealer to enter periapical tissues which can cause inflammation, leading to increased postoperative discomfort and failure of otherwise properly shaped and cleaned root canal treatment. [5],[6] Therefore, biocompatibility is as important as physical and chemical features when selecting a material for endodontic therapy. In case of endodontic sealers, studying the periapical reaction using a histological method by means of endodontic usage test (EUT) is precise and most relevant. [7],[8]

Calcium phosphate based sealers are emerging as promising candidates in endodontics because of their superior biocompatibility features. They also satisfy most of the requirements for an ideal sealer. [1],[2],[3] These materials are modified forms of self-setting calcium phosphate cements (CPC) that contain inorganic calcium and phosphate minerals, which upon wetting with an aqueous solution get converted to hydroxyapatite. [9]

The purpose of the present study is to evaluate the periapical tissue reaction to a newly developed calcium phosphate sealer, Chitra-CPC, by means of EUT using a porcine model following the International Standard ISO 7405. [10] The control used was a resin-based sealer (AH Plus).

   Materials and Methods Top


Chitra-CPC has powder and liquid components. The cement powder essentially contains tetracalcium phosphate and dicalcium phosphate dihydrate particles of size in the range of 50 μm, mixed in equimolar ratio. The cement liquid contains distilled water with Na 2 HPO 4 as the setting accelerator in an optimized concentration of 0.2 M. The net calcium-to-phosphorous ratio in the ingredients is 1.67, which leads to the formation of hydroxyapatite [Ca 10 (PO 4 ) 6 (OH) 2 ] in the set mass.

2Ca 4 (PO 4 ) 2 + 2CaHPO 4 .2H 2 O → Ca 10 (PO 4 ) 6 (OH) 2 + 4H 2 O

Chitra-CPC had already qualified (in mice for systemic response), intracutaneous reactivity test (in rabbits for skin response), pyrogen test (in rabbits for presence of pyrogens), and maximization sensitization test (in guinea pigs for allergic skin response), and muscle implantation and bone implantation tests (both in rabbits) as per the ISO 10993 criteria. [11],[12]

The reference material used in the study was a commercially available epoxy resin sealer, AH Plus (Dentsply) which is widely used clinically for endodontic obturation.


The study plan was approved by the Biological Evaluation Committee of the Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology (SCTIMST), Trivandrum. The permission to use animals was obtained from the Institutional Animals Ethics Committee, SCTIMST.

Six adult crossbred domestic pigs of either sex were randomly assigned for the study after laboratory acclimatization and were divided into two groups with three per group for 1-month (Group A) and 3-month (Group B) time periods. A total of 18 teeth were obtained in one time period. The test material CPC was used as the sealer in randomly allocated 12 teeth, whereas the reference material, AH Plus, was used in the remaining 6 teeth.

The animal implantation procedures were carried out under general anesthesia at the vivarium facility of SCTIMST under the supervision of veterinary surgeons in a strict sterile environment. The operatory and the procedures were designed in such a way so as to simulate the routine clinical situation to as near as possible. The oral cavity was kept open using a modified mouth prop. Rubber dam was applied in most of the cases and secured using dental floss and smaller size clamps. In some areas where the alignment of the tooth was confusing to the operator, rubber dam was avoided, instead of which other isolation techniques were used.

A preoperative assessment of the teeth to be operated was made using intraoral periapical radiographs and occlusal radiographs. The access preparations were made using tungsten carbide burs and diamond abrasives attached to a high-speed air rotor hand piece. The root canals were thoroughly shaped and cleaned by hand instrumentation using H-files and K-files. The canals were irrigated with 2.5% sodium hypochlorite solution and copious amounts of saline solution.

After ensuring complete debridement, the teeth were obturated with gutta-percha and sealer. Using finger pluggers with pre-determined length, the sealer was intentionally carried beyond the apex into the periapical tissues. A master cone gutta-percha was selected and the root canals were completely obturated using lateral condensation technique. The access preparations were sealed using IRM (Dentsply). After the procedure, reversal of anesthesia was done and the animals were kept under proper dose of analgesics and antibiotics and fed with soft diet. Special care was given to the animals during the period following the procedure.

The animals were euthanized at the end of the stipulated time periods, namely, 28 ± 3 days for Group A and 90 ± 5 days for Group B. The tissue which was obtained following the autopsy was fixed using 10% neutral buffered formalin for 10 days. Each treated tooth was then separated together with its surrounding hard and soft tissues in a single block using high-speed diamond precision saw. The tissue blocks were then immersed in 10% formic acid for decalcification and the bottle was continuously stirred using a magnetic stirrer for nearly 25-35 days with change of formic acid every alternate day. The blocks were removed from the solution after assuring thorough decalcification. Using a BP blade, the blocks were then sectioned parallel to the long axis of the tooth through the root canal, showing the obturated material and the periapical tissues.

The sections were processed by an extended technique using an Automatic Tissue Processor and embedded in paraffin wax for sectioning. Tissues were sectioned to 5 μm thickness using an Automatic Rotary Microtome and stained by hematoxylin and eosin. The sections were then examined by light microscopy using a binocular microscope and histological features were recorded. Images were captured using a digital camera.

For each specimen, the tissue changes were graded according to the scheme given in [Table 1].
Table 1: Scoring based on the degree of Infl ammation

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   Results Top

In the 1-month time period, CPC showed mild to moderate periapical tissue reaction [Figure 1]. CPC is lost during the decalcification process while preparing the tissues for histopathologic examination. Because of this, the areas occupied by the material are seen as large clear spaces in histological pictures [Figure 2]. Inflammatory cells are few and scattered near these clear spaces. New bone formation lined by osteoblasts is also seen. In the 11 samples assessed, mild inflammatory reaction was evident in 9 samples (score 1). The rest two samples showed a moderate type of reaction (score 2).
Figure 1: Periapical histopathologic picture of CPC sample in the 1-month time period (Group A) with ×4 magnifi cation. CPC sealer material is lost during the decalcifi cation process and the area occupied by the material is seen as voids

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In the 3-month time period, the slides of CPC mainly showed an absence of inflammation to mild inflammatory reaction in the periapical area [Figure 3]. In all the histological pictures analyzed, the clear spaces as seen in the 1-month samples were not present, indicating that the material had resorbed completely in less than 3 months duration [Figure 4]. Instead, well-healed periapical areas devoid of inflammation were observed in 2 out of the 10 samples (score 0). Mild inflammation was seen in six slides (score 1) and moderate inflammation was evident in the remaining two slides (score 2).

In the 1-month time period, predominantly severe to moderate inflammatory tissue reaction was found in the periapical tissues adjacent to AH Plus [Figure 5]. The resin sealer was demonstrable in periapical histological pictures as numerous foci of black foreign material surrounded by chronic inflammatory cells and granuloma. Out of the four slides analyzed, two had severe inflammatory reactions (score 3). One of the slides showed moderate reaction (score 2) and the other showed mild reaction (score 1).
Figure 2: Periapical histopathologic picture of CPC sample in the 1-month time period (Group A) with ×20 magnifi cation. Magnifi ed area shows the void in the periapical area originally occupied by the sealer

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Figure 3: Periapical histopathologic picture of CPC sample in the 3-month time period (Group B) with ×4 magnifi cation. Well-healed tissues devoid of infl ammation

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Figure 4: Periapical histopathologic picture of CPC sample in the 3-month time period (Group B) with ×20 magnifi cation. Note the presence of new bone formation

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Figure 5: Periapical histopathologic picture of AH Plus sample in the 1-month time period (Group A) with ×40 magnifi cation. The sealer material is demonstrable as foci of black foreign material surrounded by infl ammatory cells and macrophages

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In the 3-month time period, the slides with AH Plus showed mild to moderate periapical tissue reaction [Figure 6]. Chronic inflammatory cells, foreign body type of granuloma, and macrophages with ingested AH Plus material were observed in these slides. Out of the five samples analyzed, three slides showed mild form of reaction (score 1). One of the slides showed moderate reaction (score 2) and the remaining one showed severe inflammatory reaction (score 3).
Figure 6: Periapical histopathologic picture of AH Plus sample in the 3-month time period (Group B) with ×40 magnifi cation. Note the presence of macrophages with ingested sealer material

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The results were tabulated and Mann and Whitney "U" test was done to compare the periapical tissue reaction of the two materials [Table 2] and [Table 3].
Table 2: Scores for the one month time period

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Table 3: Scores for the three month time period

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   Discussion Top

Use of calcium phosphate preparations in dentistry in the literature dates back to 1966 when a compound of calcium phosphate, neomycin, and hydrocortisone was used for pulp capping procedures. [13] The past two decades saw numerous attempts to manufacture a calcium phosphate sealer suited for its use in endodontic therapy and tested with focus on the development of a non-mutagenic, non-carcinogenic, and an overall tissue-friendly material. [14],[15],[16]

In various biocompatibility studies, calcium phosphate preparations were found to be non-toxic, non-immunogenic, and non-mutagenic. In histological studies, when placed on fresh cut and bleeding bone, the material gets resorbed and concomitantly gets replaced by normal bone. [17] Moreover, the chemical composition of set cement resembles the bone mineral, hydroxyapatite. [18],[14]

MTT assay on L929 cells reported the absence of any cytotoxic effect with calcium phosphate sealer. [19] Human periodontal cell response to a calcium phosphate based root canal sealer (CAPSEAL I and II) revealed less cytotoxicity along with the potential to promote bone regeneration. [20] Subcutaneous tissue implantation in rats showed superior biocompatibility and less tissue irritation with calcium phosphate based root canal sealers. [21],[22],[23] When used as a perforation repair material in furcation defects in dogs, calcium phosphate preparations gave good results with healing and bone reorganization similar to Mineral Trioxide Aggregate (MTA). [24]

Similar materials were modified and used in various studies for use as pulp capping agents which showed reparative dentin formation and favorable healing of the pulp tissue. [25],[26],[27] Tricalcium phosphate was suggested for use as an apical plug, much like dentin shavings and calcium hydroxide. [28] In a more recent study, the effect of calcium phosphate cement on growth and odontoblastic differentiation in human dental pulp cells was investigated. Odontoblastic differentiation was evidenced by the formation of mineralized nodules and induction of alkaline phosphatase activity. [29]

Among all the preclinical tests available to detect the biocompatibility of a root canal sealer/filler material, EUT is the most specific and reliable. The usefulness of a usage test for predicting the biocompatibility is directly proportional to the fidelity with which the test simulates the clinical use of the material in every regard, including time, location, environment, and placement techniques. [30]

In the above animal experiment, the 1-month time period observations give a picture of the immediate reaction to the material by the host tissues. During this period, there is a definite statistically significant result, comparing the two materials, CPC and AH Plus ( P = 0.031 < 0.05) CPC definitely shows a better acceptance by the host periapical tissues in comparison with AH Plus. The reparative and regenerative capacity of living tissues to injury is quite evident in the slides examined from the 3-month group. CPC gets resorbed within a short time period of less than 3 months and shows an excellent healing of the periapical tissues. Similar experiment conducted in the teeth of monkeys also reported minimal tissue reaction and new bone formation near calcium phosphate cement when used as a sealer. [31]

Mild inflammatory reaction is evident in samples containing AH Plus in the 3-month study group. Macrophages with engulfed material are evident in these samples and eventually the material might completely resorb from the periapical area as evident from the various clinical cases showing long-term results. However, a statistically significant difference could not be found between the amounts of inflammation produced by the two materials in this time period (P = 0.211> 0.05) Even though there is no evident statistical difference in the 3-month time period between the samples, the individual histological response shows Chitra-CPC is more acceptable to the tissues. The lack of inflammation can be attributed to the cellular level reparative mechanism of the body of the animal in the course of 3 months.

The outcome of this study corroborates with the earlier results of biocompatibility evaluation of Chitra-CPC. [11],[12] The modification of the physical properties of this material can be a promise for its use in other purposes in dentistry like repair of furcal and perforation defects, root canal filler material in deciduous teeth, apexification procedures, apexogenesis, direct and indirect pulp capping, retrograde filling material, and so on.

   Conclusion Top

The materials which are introduced in any part of the human body have to be friendly to the tissues of contact and should not produce any systemic effects. EUT clearly proves the superior biocompatibility of Chitra-CPC, making calcium phosphate cements a promise to the science and practice of endodontics. Efforts continue all over the world to improve the functional efficiency of calcium phosphate biomaterials. [32] The scope of calcium phosphate material in other applications in dentistry and medicine is also worth exploring.

   Acknowledgments Top

The authors sincerely acknowledge the valuable support of Dr. P.R.Umashanker and Dr. Mira Mohanty Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Trivandrum.

   References Top

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Correspondence Address:
George M Jacob
Department of Conservative Dentistry and Endodontics, PSM College of Dental Science and Research, Akkikavu, Thrissur, Kerala
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0970-9290.131049

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]

  [Table 1], [Table 2], [Table 3]

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