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Table of Contents   
ORIGINAL RESEARCH  
Year : 2015  |  Volume : 26  |  Issue : 6  |  Page : 627-632
Histological analysis of the biocompatibility of calcium hydroxide associated with a new vehicle


1 Department of Endodontics, North of Minas Gerais Integrated College - FUNORTE/SOEBRÁS, Florianópolis, Santa Catarina, Brazil
2 Department of Multidisciplinary Clinic I and II (Endodontics), Paranaense University - UNIPAR, Francisco Beltrão, Paraná, Brazil
3 Department of Endodontics, Federal University of Uberlândia - UFU, Uberlândia, Minas Gerais, Brazil
4 Department of Pathology, Federal University of Uberlândia - UFU, Uberlândia, Minas Gerais, Brazil
5 Department of Restorative Dentistry (Endodontics), Ribeirão Preto Dental School, University of São Paulo - FORP/USP, Ribeirão Preto, São Paulo, Brazil
6 Department of Endodontics, University of São Paulo - USP/SP, São Paulo, Brazil

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Date of Submission26-Jan-2015
Date of Decision12-Jul-2015
Date of Acceptance01-Feb-2016
Date of Web Publication18-Feb-2016
 

   Abstract 

Background: Several substances have been researched to act as vehicles associated with calcium hydroxide. The specific type of vehicle is directly related to the effectiveness of the ionic dissociation, antimicrobial action, and biocompatibility of this medication.
Aim: To make a histological evaluation of the biocompatibility of calcium hydroxide associated with a new vehicle (triethanolamine), compared with polyethylene glycol, saline solution, and olive oil.
Materials and Methods: Fifty mice of guinea pig species were randomly divided into five groups (n = 10) according to each vehicle used - Group 1: calcium hydroxide, Group 2: triethanolamine, Group 3: polyethylene glycol, Group 4: saline solution, and Group 5: olive oil - and further divided into subgroups according to the two analysis periods - (a) 30 and (b) 90 days. Teflon carriers filled with the evaluated substances were placed in standardized bone cavities in the anterior mandible region. The animals were euthanized to perform a histological analysis after the time periods analyzed.
Results: In 30 days, specimens from Groups 1, 3, and 5 showed a very pronounced inflammatory response. Specimens from Group 2 showed an inflammatory reaction ranging from mild to severe, with rapid resorption of the material and progressive advancement of osteoid tissue into the teflon carriers. Specimens from Group 4 showed a moderate inflammatory reaction. In 90 days, specimens from Group 1 showed a very pronounced fibrous replacement. In regard to Group 2 specimens, the tested material was solubilized and replaced by newly formed bone tissue. For Groups 3 and 5 specimens, the inflammatory reaction went from acute to moderate. In relation to Group 4 specimens, an organized bone formation process was observed.
Conclusions: Specimens from Group 2 showed higher biocompatibility, especially as compared with the specimens from Groups 3 and 5.

Keywords: Biocompatibility, calcium hydroxide, triethanolamine

How to cite this article:
Junior JS, Machado R, de Souza CJ, Loyola AM, Vansan LP, Antoniazzi JH. Histological analysis of the biocompatibility of calcium hydroxide associated with a new vehicle. Indian J Dent Res 2015;26:627-32

How to cite this URL:
Junior JS, Machado R, de Souza CJ, Loyola AM, Vansan LP, Antoniazzi JH. Histological analysis of the biocompatibility of calcium hydroxide associated with a new vehicle. Indian J Dent Res [serial online] 2015 [cited 2019 Aug 22];26:627-32. Available from: http://www.ijdr.in/text.asp?2015/26/6/627/176929
Calcium hydroxide has been recommended for use as an intracanal medicament, because of its pronounced antimicrobial activity against most of the bacterial species found in root canal infections, and also because it has a neutralizing effect on bacterial lipopolysaccharides. Moreover, it has been used mainly to help dry persistently wet canals and to reduce inflammation and pain. It also acts as a physical barrier to prevent recontamination of root canals and helps promote tissue dissolution and limit osteo-cementum-dentin resorption, thus promoting tissue restoration through mineralization. [1],[2],[3],[4]

The efficiency of calcium hydroxide as an intracanal dressing is attributed to its ionic effect, based on chemical dissociation into calcium and hydroxyl ions, which are responsible for alkalization of the medium, resulting in a pH > 11. Its high pH induces hard tissue formation through mineralization and is also responsible for its bactericidal effect. [1],[5]

Different substances have been associated with calcium hydroxide to evaluate possible changes to its antimicrobial effects, [6],[7],[8] biocompatibility, [9],[10] pH, [5],[11],[12] ionic dissociation rate, and dentin diffusion. [12],[13],[14],[15],[16] The large number of vehicles researched and the absence of a consensus regarding the ideal vehicle are the main reasons for seeking new alternatives.

Triethanolamine is a drug that has long been used in the pharmaceutical industry because of its alkalizing potential. This characteristic enables triethanolamine to stabilize the pH of different solutions. [17],[18],[19],[20]

Therefore, the aim of this study was to make a histological evaluation of the biocompatibility of calcium hydroxide associated with triethanolamine, compared with polyethylene glycol (Calen ® ), saline solution (Calasept ® ), and olive oil (L&C paste ® ).


   Materials and Methods Top


After approval of the study by the Research Ethics Committee of the University of São Paulo - USP/SP, 50 mice of guinea pig species were selected for this study, weighing about 800 grams each. These mice were randomly divided into five groups (n = 10) according to each vehicle to be used and were further divided into subgroups according to the analysis period (30 and 90 days) [Table 1].
Table 1: Groups according to vehicle used and analysis period


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The animals received general anesthesia for inoculation of the analyzed substances by intramuscular injection of 0.6 ml of ketamine (100 mg/ml, Bukwang Pharmacological Co., Seoul, Korea) mixed with acepromazine (0.5 mg/ml, Eurofarma, São Paulo, SP, Brazil).

After incision, surgical flap deflection, and exposure of the bone tissue in the anterior mandible region, standardized cavities were prepared to accommodate the teflon carriers (Polytetrafluorethylene, DuPont, HABIA, Knivsta, Sweden) 2 mm in diameter, depth, and length; 1.3 mm inner and 2 mm outer diameters; open on one side and filled with the evaluated substances.

After placing the teflon carriers on the animals, they were euthanized by chloroform inhalation (Fórmula & Ação, São Paulo, SP, Brazil) after 30 and 90 days. Their jaws were sectioned, fixed in 10% formalin solution with a sodium cacodylate buffer at 7.4 pH (Fórmula & Ação, São Paulo, SP, Brazil), and kept 48 h at 4°C for histological prefixation. Then, the plates were washed for 12 h in running water and decalcified with 5% aqueous nitric acid (Fórmula & Ação, São Paulo, SP, Brazil).

The dehydration process was performed using increasing concentrations of ethyl alcohol solution: 50%, 70%, 90%, and absolute (Fórmula & Ação, São Paulo, SP, Brazil). Afterward, the specimens were placed in paraffin to make 24 blades for each group, resulting in 120 histological semi-serial slices, about 5 μm thick, stained with hematoxylin and eosin for analysis by light microscopy.

Tissue response evaluation was based on qualitative and semi-quantitative observations of any phenomenon related to cell and tissue inflammation and repair found immediately at or at a distance from the teflon carrier/tissue interface. The type and severity of the inflammatory infiltrate were determined by two independent observers who had been calibrated for the presence or absence of (i) neutrophils, (ii) macrophages, (iii) lymphocytes, (iv) plasmocytes, (v) foreign body giant cells, (vi) dispersed material, (vii) capsules, (viii) new bone formation, (ix) tissue necrosis, and (x) bone resorption. These features were scored for intensity: (+) Absent or mild, (++) moderate, and (+++) intense. [21],[22]


   Results Top


The quantification of cells involved in the inflammatory process, the different histopathological features [Table 2], and the description of the histological images are described below.
Table 2: Qualitative and semi-quantitative characterization of the inflammatory process at 30 and 90 days, after intraosseous implants of the materials, based on Fédération Dentaire International and American Dental Association recommendations


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The specimens from Groups 1a, 3a, and 5a showed a very pronounced inflammatory response.

The specimens from Group 2a showed an inflammatory reaction ranging from mild to severe, with rapid resorption of the material and progressive advancement of osteoid tissue into the teflon carriers, the presence of cells of chronic inflammation (macrophages, lymphocytes, and giant cells), hyperemia, and dispersed material in the intimacy of the newly formed tissue [Figure 1].
Figure 1: Light micrographs of sections of a specimen from Group 2a, showing an inflammatory reaction ranging from mild to severe, with rapid resorption of the material and progressive advancement of osteoid tissue into the teflon carriers, the presence of cells of chronic inflammation (macrophages, lymphocytes, and giant cells), hyperemia, and dispersed material in the intimacy of the newly formed tissue (a) ×110; (b) ×120; (c) ×230; (d) ×250

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The specimens from Group 4a showed a moderate inflammatory reaction, with a neutrophilic infiltration occupying the teflon carriers almost entirely [Figure 2].
Figure 2: Light micrographs of sections of a specimen from Group 4a, showing moderate inflammatory reaction with neutrophilic infiltration occupying the teflon carriers almost entirely (a) ×110; (b) ×120; (c) ×230; (d) ×250

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In specimens from Group 1b, there was no typical fibrous capsule but very pronounced fibrous replacement [Figure 3].
Figure 3: Light micrographs of sections of a specimen from Group 1b, showing very pronounced fibrous replacement (a) ×110; (b) ×120; (c) ×230; (d) ×250

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In all specimens from Group 2b, the tested material was solubilized and replaced by newly formed bone tissue. Foreign body giant cells were also seen permeating the newly formed bone interface/teflon carriers in almost the entire extension, indicating an inflammatory reaction of low intensity.

The inflammatory reaction of the specimens from Groups 3b and 5b went from acute to moderate [Figure 4].
Figure 4: Light micrographs of sections of a specimen from Groups 3b (a and b) and 5b (c-f), showing an inflammatory reaction ranging from acute to moderate (a) ×110; (b) ×120; (c) ×110; (d) ×120; (e) ×110; (f) ×120

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An organized bone formation process without fibrous capsules was observed in specimens from Group 4b [Figure 5].
Figure 5: Light micrographs of sections of a specimen from Group 4b, showing an organized bone formation process without fibrous capsules. (a) ×110; (b) ×120; (c) ×230; (d) ×250

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The specimens from Group 5b showed replacement of the fibrous tissue by newly formed connective tissue and reduction in the inflammatory reaction. However, there was no new bone formation.


   Discussion Top


In view of the anatomical complexity and the resistance of some microbial species present in the root canal system, some studies have pointed to the need to use an intracanal dressing mainly to reduce this microbial contingent and its subproducts to levels that favor healing. [23],[24] Since calcium hydroxide is the most widely used intracanal dressing worldwide and its effects are associated with the vehicle used, [5],[6],[7],[8],[12],[14] the lack of a consensus on the ideal vehicle warrants the search for new alternatives.

Insofar, as one of the main requirements of new materials and substances, is potentially low cytotoxic effect on tissues; [25],[26],[27] the aim of this study was to make a histological evaluation of the biocompatibility of calcium hydroxide associated with a new vehicle (triethanolamine), compared with traditional formulations, such as Calen ® , Calapset ® , and L&C paste ® .

In general, biocompatibility tests carried out by histological analyses aim at qualifying the inflammatory reactions occurring between tissues and evaluated materials. [9],[26],[28] However, these reactions are "three-dimensional processes" and a numerical rating may be dubious because of the two-dimensionality permitted by this type of analysis. For this reason, detailed descriptions and semi-quantitative analysis of inflammatory processes based on Fédération Dentaire Internationale and American Dental Association recommendations [21],[22] [Table 2] were performed. These descriptions were carried out by two completely blinded evaluators, to obtain total impartiality of the results. [28]

The specimens from Group 2 showed less aggressive inflammatory processes than the specimens from the other groups in both study periods (30 and 90 days). This conclusion is based on the histological observations made, mainly being the resorption of the material and a marked, progressive growth of bone tissue in the teflon carriers, with different levels of maturation and specific cells of chronic inflammation (macrophages, lymphocytes, and giant cells).

Most likely, these results are associated with the chemical composition of triethanolamine, an organic compound considered both tertiary amine and tri-alcohol (C 6 H 15 NO 3 ) with a low degree of toxicity. Triethanolamine acts as a weak base, owing to the lone pair of electrons on the nitrogen atom, and has excellent potential for balancing pH when in contact with different solutions. [17],[18],[19],[20] Even though the pH of the solutions used in this study was not evaluated, triethanolamine was probably responsible for balancing the pH of the calcium hydroxide paste used as a vehicle, based on the less intense inflammatory processes observed histologically in both study periods (30 and 90 days). Similar results were observed when using saline solution as a vehicle (Group 4).

Guerreiro-Tanomaru et al. [16] performed a study showing that the association between calcium hydroxide and saline solution showed a progressive increase in pH values up to 14 days but remained unchanged at 21, 30, and 60 days. This may be attributed to the reduction in the available number of hydroxyl ions because of the rapid ionic dissociation that occurs when using an aqueous vehicle. [29] This also may explain the good results for the biocompatibility of the specimens from Groups 4a and b.

On the other hand, calcium hydroxide associated with oily vehicles, such as polyethylene glycol and olive oil (Groups 3 and 5, respectively), is capable of releasing the hydroxyl ions more slowly and causing potential irritation to the tissues for a longer period. [30]

Since the basic component of all pastes analyzed was calcium hydroxide, the variation in the tissue response observed may be attributed to the substances added to the pastes. The comparison of the tissue responses evaluated in this study led the authors to conclude that calcium hydroxide associated with triethanolamine was the most biocompatible association, followed by Calasept ® , Calen ® , and L&C paste ® . However, it should be borne in mind that these conclusions are based on the evaluation of tissue reactions using an animal model, the results of which cannot necessarily be extrapolated to those of apical and periapical tissues. However, it is important to conduct future evidential studies with similar methodological designs to prove the biocompatibility of calcium hydroxide associated with triethanolamine, and later studies aiming specifically at evaluating the pH of this association, and its antimicrobial effects on several types of microorganisms.


   Conclusions Top


Calcium hydroxide paste associated with triethanolamine showed higher biocompatibility, especially compared with Calen ® and L&C paste ® .

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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Correspondence Address:
Prof. Ricardo Machado
Department of Multidisciplinary Clinic I and II (Endodontics), Paranaense University - UNIPAR, Francisco Beltrão, Paraná
Brazil
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-9290.176929

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