doi:10.1111/iej.12328
Biocompatibility of root filling pastes used in primary teeth
nior2, M. S. Rizzo3, R. D. Moura4, M. S. Moura5, M. D. M. C. C. B. Lima1, A. M. Conde Ju 5 5 Lima & L. F. A. D. Moura 1
Postgraduate Programme in Dentistry, Federal University of Piauı (UFPI), Teresina; 2Department of Morphology, Federal University of Piauı (UFPI), Teresina; 3Department of Clinic and Veterinary Surgery, Federal University of Piauı (UFPI), ao Paulo (USP), S~ ao Paulo; and 5Department of Pathology Teresina; 4Postgraduate Programme in Pathology, University of S~ and Dentistry Clinic, Federal University of Piauı (UFPI), Teresina, Brazil
Abstract nior AM, Rizzo MS, Moura RD, Lima CCB, Conde Ju Moura MS, Lima MDM, Moura LFAD. Biocompatibility of root filling pastes used in primary teeth. International Endodontic Journal.
Aim To evaluate the biocompatibility of two pastes designed to fill the root canals of primary teeth. Methodology A study group of 54 mice received subcutaneous tissue implants of polyethylene tubes containing CTZ or calcium hydroxide paste or, as a negative control, empty tubes. Biocompatibility was evaluated on days 7, 21 and 63, yielding a total of nine groups of six animals each. Following the experimental intervals, the implant areas were removed and subjected to histologic processing. After the tissues were stained with HE and Masson trichrome, two pathologists performed a histologic analysis of the samples in a blinded manner. Collagen fibre formation, tissue thickness and inflammatory cell infiltration were analysed qualitatively. Quantitative morphometry was performed for the thickness, perimeter length and tissue
Introduction The complex morphology of root canal systems and the root resorption of primary molars are uncontrollable factors that may hinder proper chemical–mechanical
Correspondence: L ucia de F atima Almeida de Deus Moura, Department of Pathology and Dentistry Clinic, Federal University of Piauı, Rua Angelica, 1650, Bairro de Fatima, CEP 64049-532, Teresina, Piauı, Brazil (Tel.: +55 86 3233 3050; e-mail: [email protected]).
© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
area of the region in direct contact with the open tube. with the Tukey post-test and Kruskal–Wallis analysis followed by Dunn’s post-test, with significance established as P < 0.05, were used for data analysis. Results At 7 days, all groups had severe acute inflammatory infiltrates. Inflammation was reduced at 21 days in the CTZ paste group. Mild chronic inflammatory infiltrates were observed after 63 days in the CTZ and Ca(OH)2 paste groups; these groups also showed a significant decrease in collagen fibre density (P < 0.05), which was not observed in the control group. The average tissue thickness, perimeter length and area in contact with the tube decreased during the experimental periods in all groups. Conclusion The CTZ and calcium hydroxide pastes demonstrated biocompatibility with subcutaneous tissue in this experimental model. ANOVA
Keywords: biocompatibility, calcium hydroxide, primary teeth, root canal filling materials, subcutaneous tissue. Received 26 August 2013; accepted 29 May 2014
canal disinfection in primary teeth with pulp necrosis (Harini Priya et al. 2010, de Sousa et al. 2011). These conditions may also impede the development of permanent tooth follicles as well as the establishment of accurate working length (Molander & Dahlen 2003, Barja-Fidalgo et al. 2011, Gondim et al. 2012). Most of the recommended techniques for filling root canals of primary teeth propose the use of instrumentation and the chemo-mechanical preparation of the root canal system (American Academy of Pediatric
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Dentistry 2012). The use of calcium hydroxide (Ca (OH)2) paste predominates over other techniques for the pulp therapy of primary teeth. As a root filling material, Ca(OH)2 paste demonstrates antibacterial action, biocompatibility and tissue repair capacity (Hauman & Love 2003b, Mohammadi & Dummer 2011). Alternative pulp therapies that have been developed include those that recommend restricting treatment to the pulp chamber and methods that may promote the disinfection or sterilization of root canals with necrotic pulps, as in the use of antibiotic-containing pastes (Cappiello 1964, Takushige et al. 2004, Pinky et al. 2011, Trairatvorakul & Detsomboonrat 2012). Cappiello (1964) developed a paste for filling root canals in primary teeth. The paste comprises chloramphenicol, tetracycline and zinc oxide with eugenol as a vehicle and is currently known as CTZ paste (powder base provided by a pharmacy in proportions of 1 : 1 : 2 chloramphenicol/tetracycline/zinc oxide, respectively). This method obviates the need for canal instrumentation, thereby facilitating its use in young children and users of public health services (Cappiello 1964). Successful endodontic treatment of teeth with pulp necrosis requires reducing or eliminating infection within the root canal system. The medicaments used in the pulp therapy of primary teeth should possess antimicrobial activity and be biocompatible, particularly given the proximity of contact in the furcation and periapical regions (Silva et al. 2010, Barja-Fidalgo et al. 2011). Biocompatibility is the ability of a material that is in contact with a tissue to promote an appropriate biological response with minimal inflammatory reactions. Methods that deploy substances in the subcutaneous tissues of laboratory animals are commonly used to evaluate the biocompatibility of endodontic materials (Hauman & Love 2003a, Viola et al. 2012). The purpose of this study was to evaluate the biocompatibility of two pastes for the root canal treatment of primary teeth (CTZ and calcium hydroxide pastes). The null hypothesis was that there is no difference between the biocompatibility of CTZ and calcium hydroxide pastes in mouse subcutaneous tissue.
Materials and methods Ethical approval The procedures used in this study were in accordance with a protocol reviewed and approved by the Ethics Committee on Animal Experimentation, Federal
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University of Piauı (UFPI) (opinion No. 067/12) and with the ethical guidelines for animal experimentation advocated in the ‘European Communities Council Directive’ of 24 November 1986 (86/609/EEC) regarding recommendations for the care and use of laboratory animals. This experimental trial began in November 2012 and was concluded in May 2013 upon euthanasia of the last test animal.
Animal model A total of 54 mice (Mus musculus) were used. The animals were Swiss albino males from 6 to 8 weeks of age that weighed between 15 and 30 g. Throughout the experiment, the animals were housed in a vivarium with standard rations and access to water ad libitum. All animals were examined every 2 days and monitored for local or systemic changes. The animals were wormed with Baskenâ suspension (14.5 mg pyrantel pamoate + 9.5 mg oxantel pamoate kg 1), which was administered orally in a single dose 7 days prior to the onset of the experimental procedural.
Root canal pastes The biocompatibility of the CTZ and calcium hydroxide pastes was tested using an empty tube as a negative control. Histological analysis was performed on the tissue samples for each material at three different intervals: 7, 21 and 63 days. The tissue response was analysed for nine groups with six animals per group, which were distributed as follows: Group I – six animals received a polyethylene tube containing CTZ paste for 7 days; Group II – six animals received a polyethylene tube containing CTZ paste for 21 days; Group III – six animals received a polyethylene tube containing CTZ paste for 63 days; Group IV – six animals received a polyethylene tube containing calcium hydroxide paste for 7 days; Group V – six animals received a polyethylene tube containing calcium hydroxide paste for 21 days; Group VI – six animals received a polyethylene tube containing calcium hydroxide paste for 63 days; Group VII – six animals received an empty polyethylene tube for 7 days; Group VIII – six animals received an empty polyethylene tube for 21 days; and Group IX – six animals received an empty polyethylene tube for 63 days. The micronized powder base for the CTZ paste was provided in 250-mg capsules by a compounding
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pharmacy (Teresina, PI, Brazil), and the paste comprised chloramphenicol 62.5 mg, tetracycline 62.5 mg and zinc oxide 125 mg with 0.1 mL of eugenol vehicle (Biodin^ amica, Ibipor~a, PR, Brazil). Calcium hydroxide (Ca(OH)2) powder (analytical grade) (Biodin^ amica) was provided in 250-mg capsules and reconstituted in 0.2 mL of distilled water (Isofarma, Eusebio, CE, Brazil). To prepare the two pastes, the powders were dispensed onto sterile glass plates and mixed with their respective vehicles by a metal spatula at the time of use.
Experimental design for subcutaneous implantation Animals were pre-medicated with Tramadol at a dose of 2 mg kg 1 (Tramadol hydrochloride 50 mg mL 1, Hipolaborâ; Sabar a, MG, Brazil) by deep intramuscular injection with a 1-mL syringe. After 10 minutes, anaesthesia was induced using a combination of xylazine (10 mg kg 1) (xylazine 2% Xilazin â; Syntec, Cotia, SP, Brazil) and ketamine (80 mg kg 1) (ketamine hydrochloride 50 mg mL 1, Clortaminaâ; BioChimico, Itatiaia, RJ, Brazil) prepared in the same syringe and administered by deep intramuscular injection. The back of the animal was shaved, and the surgical region was disinfected with a 1% solution of chlorhexidine gluconate. In preparation for the dorsal implantation in each animal, polyethylene tubes that were 1 mm in internal diameter (peripheral catheter type scalp No. 27 Solidorâ; Shijiazhuang, China) were sterilized in ethylene oxide and closed at one end with an Allis forceps. The tubes were heated, sliced into 1-cm segments and filled with the material to be tested. After a 0.5-cm linear incision was made at the caudal lumbar dorsum, the soft tissue was dissected, and a tube was inserted into the tissue. The skin incision was closed with 5.0 nylon sutures (Shalonâ Sutures; S~ ao Luis de Montes Belo, GO, Brazil). For the first 24 h following implantation, the animals received tramadol analgesia (2 mg kg 1), which was administered by deep intramuscular injection every 6 h. At the end of 7, 21 or 63 days, animals from each group were pre-medicated with tramadol (2 mg kg 1) by deep intramuscular injection and euthanized with an anaesthetic combination of xylazine (10 mg kg 1) and ketamine (80 mg kg 1) prepared in the same syringe and administered by deep intramuscular injection. The implant area, which comprised polyethylene
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tubing, subcutaneous tissue and skin, was removed and fixed in buffered 4% paraformaldehyde for 24 h.
Histologic analysis Following fixation, the polyethylene tubes were removed from the fixative, dehydrated in increasing concentrations of alcohol, cleared in xylene, embedded in paraffin and blocked. The blocks were cut using a microtome (Leica RM 2245) to yield 5-lmthick longitudinal semi-serial sections. Tissues were stained with haematoxylin and eosin (HE) and Masson trichrome (Putt 1972). The sections were examined under a binocular light microscope (Olympus, Tokyo, Japan) at 100, 200 and 4009 magnification and digitally photographed. A histologic analysis was performed by two pathologists, who were blind to the experimental conditions, at different times. Both examiners analysed the same fields. The Masson trichrome and HE stains were used to analyse the collagen fibre formation and thickness, respectively, at 2009 magnification. The inflammatory infiltrate was analysed at 4009 magnification with HE stain. The intra-examiner agreement was based on two histologic evaluations performed at different intervals on 10% of the sections, which were arranged in order from the smallest to the largest increase (j = 0.80 and 0.81). The interexaminer agreement (j = 0.79) indicated interexaminer reliability. The tissues that contacted the open end of the tubes were analysed and graded for the following histologic findings: 1. formation of collagen fibres, 2. tissue thickness and 3. inflammatory infiltrate. The density of collagen fibre formation was graded as 0 (absent), 1 (mild – collagen fibres arranged similarly to normal connective tissue), 2 (moderate – individual collagen fibres were observed, but did not display linear features or a typical wavy microanatomy) and 3 (severe – very dense, individual collagen fibres cannot be distinguished) (Silva et al. 2009, Queiroz et al. 2011). The tissue thickness was assessed by the extent of fibroblast cell layers formed and the presence of macrophages in the peripheral contact region of the tube and classified as follows: 0 (normal – no layer), 1 (slightly increased – 1 to 3 layers), 2 (moderately increased – 4 to 10 layers) and 3 (intensely increased – more than 10 layers). The inflammatory infiltrate was analysed qualitatively to assess the characteristics and concentration of polymorphonuclear cells (PMN) and mononuclear cells, which were classified as
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follows: 0 (no cells), 1 (mild – 1 to 10 cells), 2 (moderate – 11 to 20 inflammatory cells) and 3 (intense – inflammatory cell concentration ≥21 cells) (Silva et al. 2009, Queiroz et al. 2011).
Morphometric analysis Morphometry, via quantitative analysis of the tissue area that directly contacted the open part of the tube, was performed using imaging software (Leica QWin – Image Processing and Analysis Software Leica D1000, version 4.1; Cambridge, UK). Estimated values for the tissue thickness (lm), perimeter (lm) and area (lm2) were obtained by image analysis of the photomicrographs.
Statistical analysis Data were tabulated and analysed using SPSS version 18.0 (SPSS Inc., Chicago, IL, USA). The Shapiro–Wilk test was used to test the null hypothesis. ANOVA with the Tukey post-test was used to verify differences between the areas and perimeter thicknesses, and the data grading scores were analysed by the Kruskal– Wallis test followed by Dunn’s post-test, with significance established as P < 0.05 (Fig. 1).
Results Upon removal, all implants were covered with a visible, palpable and transparent fibrous capsule. There were no signs of rejection in the specimens. The tubes containing CTZ paste displayed progressive darkening over time. The results from the qualitative histologic analysis of the collagen fibre formation, thickness and inflammatory infiltration of connective tissue adjacent to the open ends of the polyethylene tubes are presented in Table 1 and Figs 2, 3 and 4. When the same treatment was compared for different experimental periods (7, 21 and 63 days), significant differences were observed in the reduction of collagen fibre formation (P < 0.05) and inflammatory cell infiltration (P < 0.05) for the specimens that were treated with calcium hydroxide paste. In the group implanted with an empty tube, increased formation of collagen fibres (P < 0.05) was observed, and no significant difference was observed with the CTZ paste at different times (P > 0.05). The mean values with standard deviations for the thickness (lm), perimeter (lm) and area (lm2) of the reactive tissue in direct contact with the open tubes are presented in Table 2.
Figure 1 Study flowchart.
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Moderately increased (Fig. 3a) Mild to moderately increased (Fig. 3d) Severely increased (Fig. 3g)
Mild collagen fibre density (Fig. 2d) Mild collagen fibre density (Fig. 2g)
Severe; mononuclear and occasional neutrophils present (Fig. 4c) Mild; predominantly mononuclear with occasional neutrophils (Fig. 4f) Mild; predominantly mononuclear with occasional neutrophils (Fig. 4i)
Moderate; predominantly mononuclear, with the possible presence of neutrophils (Fig. 4e) Moderate; with macrophages, neutrophils and neovascularization (Fig. 4h)
Moderately increased (Fig. 3c) Mild to moderately increased (Fig. 3f) Slightly increased (Fig. 3i)
Collagen fibres admixed with extracellular matrix without any typical linear or wavy forms; individual collagen fibres could not be distinguished (Fig. 2c) Moderate (Fig. 2f) Mild to moderate (Fig. 2i)
63c
Severe; presence of macrophages, lymphocytes, neutrophils and newly formed vessels (Fig. 4b)
Severely increased (Fig. 3b) Slightly increased (Fig. 3e) Moderately increased, marked presence of fibroblasts (Fig. 3h)
Mild collagen fibre density (Fig. 2e) Individual collagen fibres observed as in normal connective tissue (Fig. 2h)
Individual collagen fibres and alternating extracellular matrix without linear features or wavy fibres (Fig. 2b)
21b
Experimental period (days)
P: Kruskal–Wallis. a There was no statistically significant difference between groups (P > 0.05). b There was no statistically significant difference between groups (P > 0.05). c There was a statistically significant difference between the groups in the collagen fibre formation during this period (P < 0.05).
Inflammatory infiltrate (Fig. 4a–j) Empty tube Severe; presence of lymphocytes, macrophages, neutrophils, and newly formed vessels (Fig. 4a) Calcium hydroxide paste Moderate; mixed macrophages and neutrophils present (Fig. 4d) CTZ paste Severe; predominantly mononuclear, with neutrophils present (Fig. 4g)
Tissue thickness (Fig. 3a–j) Empty tube Calcium hydroxide paste CTZ paste
Calcium hydroxide paste CTZ paste
Formation of collagen fibres (Fig. 2a–j) Empty tube Mild to moderate (Fig. 2a)
Material tested
a
Table 1 Qualitative histologic analysis of the collagen fibre formation, thickness and inflammatory cell infiltration of the connective tissue adjacent to the open ends of the polyethylene tubing
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Ca(OH)2 paste
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Figure 2 (a–i) Photomicrographs stained with Masson trichrome showing the formation of collagen fibres in the connective tissue adjacent to the open ends of the polyethylene tubes in contact with the empty tube (a, d, g), calcium hydroxide paste (Ca (OH)2) (b, e, h) and CTZ paste (c, f, i), at 7, 21 and 63 days, respectively (2009 magnification). (j) Groups for the qualitative analysis of the collagen fibre formation. (1) Space occupied by the tube and (2) subcutaneous tissue.
Discussion A great challenge facing Paediatric Dentistry is to develop biomaterials that possess antimicrobial properties, in addition to being biocompatible and resorb-
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able, for primary teeth with necrotic pulps (Silva et al. 2010, Barja-Fidalgo et al. 2011, Queiroz et al. 2011). CTZ paste has great potential as a treatment option for endodontically compromised primary teeth in
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Lima et al. Materials biocompatibility
Empty tube
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Figure 3 (a–i) Photomicrographs showing the HE staining for the tissue thickness adjacent to the open end of polyethylene tube in contact with the empty tube (a, d, g), calcium hydroxide paste (Ca(OH)2) (b, e, h) and CTZ paste (c, f, i), at 7, 21 and 63 days, respectively (2009 magnification). (j) Groups for the qualitative analysis of tissue thickness. (1) Space occupied by the tubing, (2) subcutaneous tissue, (*) neovascularization, (f) fibroblasts, (l) lymphocytes, (m) macrophages and (n) neutrophils.
© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
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Empty tube
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Figure 4 (a–i) Photomicrographs of HE-stained tissue showing inflammatory infiltrate in the connective tissue adjacent to the open end of the polyethylene tube in contact with the empty tube (a, d, g), calcium hydroxide paste (Ca(OH)2) (b, e, h) and CTZ paste (c, f, i), at 7, 21 and 63 days, respectively (4009 magnification). (j) Groups for the qualitative analysis of the inflammatory reaction. (1) Space occupied by the tubing, (2) subcutaneous tissue, (*) neovascularization, (f) fibroblasts, (l) lymphocytes, (m) macrophages and (n) neutrophils.
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Table 2 Mean values with standard deviations for thickness (lm), perimeter (lm) and area (lm2) of tissue reaction
Thickness (lm) CTZ Ca(OH)2 Control P Perimeter (lm) CTZ Ca(OH)2 Control P Area (lm2) CTZ Ca(OH)2 Control P
Day 7 M (SD)
Day 21 M (SD)
Day 63 M (SD)
135.9 (59.0) 84.7 (17.2) 108.3 (41.6) 0.091
121.4 (69.6) 92.0 (28.5) 87.1 (22.6) 0.389
88.8 (41.0) 76.4 (26.1) 70.4 (31.8) 0.634
0.376 0.552 0.121
1046.1 (252.3) 884.4 (253.8) 1079.6 (73.1) 0.261
950.0 (211.8) 894.3 (143.0) 880.4 (115.2) 0.738
941.2 (179.0) 916.7 (173.4) 763.8 (190.9) 0.218
0.656 0.958 0.004
53554.9 (16442.2) 32132.6 (11382.7) 49362.2 (9999.3) 0.027
47344.8 (27953.0) 32404.8 (9832.4) 32561.9 (12713.6) 0.308
33816.3 (13998.1) 24996.0 (1335.6) 23483.9 (13930.3) 0.396
0.261 0.473 0.008
P*
M = mean; SD = standard deviation. P: ANOVA test, applied between the materials in same experimental period. * P: ANOVA test, applied for each material between the different experimental periods.
young children with behavioural problems. Another application for CTZ paste is for users of public health services because of the large patient loads in such facilities and the ease with which general practitioners can perform these procedures due to the technical simplicity, excellent clinical outcomes and cost-effectiveness (Cappiello 1964, Bruno et al. 2007). There is a lack of scientific literature on the use of CTZ paste in clinical settings. This study is part of a macro-development project that encompasses research on the performance and antimicrobial action of CTZ paste and its components. Biocompatibility is the ability of an applied material or substance to trigger the intended response in the host. That is, although a biocompatible material may not be inert, the reaction caused by a test material must not pose an unacceptable physiological hazard when compared with other scientifically approved materials (Wataha 2001, Peters 2013). Three tests are recommended for evaluating the biocompatibility of endodontic materials: initial, secondary and application. The method used in this study was a sub-type that is used to evaluate biological tissue responses around the test material and to compare such results to biocompatible controls with established scientific acceptability (Olsson et al. 1981, ISO 7405 1996, ISO 10993-6 2007a, ISO 10993-12 2007b, Hauman & Love 2003a, Silva et al. 2009, Pereira et al. 2012). Subcutaneous implants, which were introduced by Torneck (1966), are used most commonly in dentistry.
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Compatibility with living tissue in the periapical or interradicular region is one of the most important properties for materials used in pulp therapy of primary teeth due to the occurrence of root resorption and the presence of anatomical variations that may jeopardize periodontal and periapical tissues, which may in turn endanger the development of permanent teeth (Lacativa et al. 2012). Polyethylene tube implants are used experimentally to simulate the relationship between the tooth apex and periapical tissues because the material within the tubes diffuses in a restricted area (Olsson et al. 1981, International Organization for Standardization 2007a, Silva et al. 2009, Khashaba et al. 2011, Marques et al. 2011, Mutoh & Tani-Ishii 2011). In this study, the groups in which empty polyethylene tubes were implanted had higher scores in the qualitative histologic analysis for fibroplasia and had significant differences in the perimeter and area of the tissue regions in direct contact with the open tube. This result was expected because no materials were introduced that could induce an acute inflammatory response or reaction. However, other studies have reported responses to empty tubes, albeit less intense than those observed for the experimental parameters analysed at the time (Silva et al. 2009, Queiroz et al. 2011). Significant reductions were observed in the density of collagen fibres and the inflammatory reaction when testing the calcium hydroxide paste for different experimental periods. Calcium hydroxide is widely
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used in dentistry due to its biocompatible properties and antimicrobial, anti-inflammatory and reparative activities (Hauman & Love 2003a,b, Mohammadi & Dummer 2011, de Sousa et al. 2011, Gondim et al. 2012). The biocompatibility results obtained in this study for calcium hydroxide paste are consistent with those reported by several authors (Holland et al. 2001, Semenoff et al. 2008, Silva et al. 2010, Andolfatto et al. 2012). In the present study, the tubes containing CTZ paste displayed progressive darkening over time, which may have been caused by the tetracycline component. The CTZ paste was associated with an initial intense inflammatory response during the first days of the experiment. At the end of the experiment, a mild chronic inflammatory process was observed, with a qualitative and quantitative decrease in the density of collagen fibres and tissue thickness. This finding suggests that CTZ was biocompatible and showed therapeutic properties. In a previous study, histopathologic analysis of pulpotomy performed in dogs’ teeth treated with CTZ paste demonstrated the occurrence of an initial intense inflammatory process in the coronal pulp with reduced inflammation at the end of the experiment (Bruno et al. 2007). However, the literature lacks studies on the biocompatibility of CTZ paste; thus, it was not possible to compare the results obtained in this study with those of other published studies. The normal reaction to any tissue injury tends to manifest as an acute neutrophilic inflammatory infiltrate (Serhan 2010). Given that acute histopathologic tissue behaviour is similar across all groups, studies that aim to test the biocompatibility of a drug that may cause a tissue reaction should be focused on chronic inflammatory reactions. In this study, the pastes and the control group showed variable chronic inflammatory processes at different experimental times. A histologic hallmark of successful pulp therapy is the development of reparative tissue (Torneck 1966). In the present study, a significant difference in the density of collagen fibres was observed between the two pastes at the end of the experiment. The difference was characterized by a decrease in the collagen fibres in specimens containing either the CTZ or the calcium hydroxide paste compared with the negative control. This finding is important because the two pastes behaved in manner that favours tissue repair. The results are consistent with other studies that reported similar methodologies (Sousa et al. 2004, Batista et al.
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2007, Silva et al. 2010, Marques et al. 2011, GomesFilho et al. 2012). Other materials are used as pastes for the root canal treatment of primary teeth, including pastes containing zinc oxide and eugenol (ZOE) with iodoform or a paste that combines iodoform and calcium hydroxide (Ranly & Garcia-Godoy 2000, American Academy of Pediatric Dentistry 2012). Studies with ZOE as a root canal filling material reported a chronic inflammatory reaction and slow resorption in relation to the tooth (Fuks 2000, Hauman & Love 2003b). The iodoform pastes, when combined with calcium hydroxide, exhibit good biological response and antimicrobial properties and are more easily reabsorbed at the periapical region (Ranly & Garcia-Godoy 2000). However, the techniques proposed for using these materials recommend the use of instrumentation and chemical–mechanical systems for the root canals of primary teeth (American Academy of Pediatric Dentistry 2012). The limitations of this study are inherent to preclinical experimental studies. Although the results presented here do not reflect a complete analysis of the reactions that occur in the pulp, periapical or interradicular regions, the results are important for the preliminary assessment of the irritative potential of the tested materials and subsequent biological responses.
Conclusion The initial inflammatory reactions in mice induced by CTZ and calcium hydroxide pastes regressed during the experimental periods; in addition, these pastes resulted in less-dense fibrous connective tissue, compared with the results using the empty control. These reactions support the biocompatibility of the two pastes.
Acknowledgements This study was supported financial by a scholarship grant from the Coordination of Improvement of Higher Education Personnel (CAPES).
Author contributions CCBL participated in the project design and data collection and analysis and interpretation of data and wrote the article. AMCJ participated in the project design and data collection and analysis and interpretation of data and wrote the article. MSR
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participated in the analysis and interpretation of data and wrote the article. RDM participated in the analysis of data. MSM revised the article. MDML participated in the study preparation and revised the article. LFADM conceived the study, participated in the study preparation and coordination, and wrote the article. All of the authors read and approved the final manuscript.
References American Academy of Pediatric Dentistry (2012) Guideline on pulp therapy for primary and young permanent teeth. Pediatric Dentistry 34, 222–9. Andolfatto C, da Silva GF, Cornelio AL et al. (2012) Biocompatibility of intracanal medications based on calcium hydroxide. ISRN Dentistry 2012, 1–6. Barja-Fidalgo F, Moutinho-Ribeiro M, Oliveira MAA, Oliveira BH (2011) A systematic review of root canal filling materials for deciduous teeth: is there an alternative for zinc oxide-eugenol? ISRN Dentistry 1, 1–7. Batista RF, Hidalgo MM, Hernandes L et al. (2007) Microscopic analysis of subcutaneous reactions to endodontic sealer implants in rats. Journal of Biomedical Materials Research Part A 81, 171–7. Bruno GB, Alves APN, Menezes VA, Maia MCG, Bruno JA, Viana GSB (2007) Biocompatibility evaluation of an antibiotic paste after pulpotomy in dogs. Brazilian Journal of Oral Sciences 6, 1397–401. Cappiello J (1964) Tratamentos pulpares em incisivos prim arios. Revista de la Asociacion Odontologica Argentina 52, 139–45. Fuks AB (2000) Pulp therapy for the primary and young permanent dentitions. Dental Clinics of North America 3, 571–96. Gomes-Filho JE, Duarte PC, de Oliveira CB et al. (2012) Tissue reaction to a triantibiotic paste used for endodontic tissue self-regeneration of nonvital immature permanent teeth. Journal of Endodontics 38, 91–4. Gondim JO, Avaca-Crusca JS, Valentini SR, Zanelli CF, Spolidorio DM, Giro EM (2012) Effect of a calcium hydroxide/ chlorhexidine paste as intracanal dressing in human primary teeth with necrotic pulp against Porphyromonas gingivalis and Enterococcus faecalis. International Journal of Paediatric Dentistry 22, 116–24. Harini Priya M, Bhat SS, Sundeep Hegde K (2010) Comparative evaluation of bactericidal potential of four root canal filling materials against microflora of infected non-vital primary teeth. Journal of Clinical Pediatric Dentistry 35, 23–9. Hauman CHJ, Love RM (2003a) Biocompatibility of dental materials used in contemporary endodontic therapy: a review. Part 1. Intracanal drugs and substances. International Endodontic Journal 36, 75–85.
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Hauman CHJ, Love RM (2003b) Biocompatibility of dental materials used in contemporary endodontic therapy: a review. Part 2. Root-canal-filling materials. International Endodontic Journal 36, 147–60. Holland R, de Souza V, Nery MJ et al. (2001) Reaction of rat connective tissue to implanted dentin tube filled with mineral trioxide aggregate, Portland cement or calcium hydroxide. Brazilian Dental Journal 12, 3–8. International Organization for Standardization (1996) 7405. Dentistry – Preclinical evaluation of biocompatibility of medical devices used in dentistry – Test methods for dental materials. Geneva, Switzerland: International Standardization Organization. International Organization for Standardization(2007a) 109936. Biological evaluation of medical devices. Part 6. Tests for local effects after implantation. Geneva, Switzerland: International Standardization Organization. International Organization for Standardization (2007b) 10993-12. Biological evaluation of medical devices Part 12: Sample preparation and reference materials. Geneva, Switzerland: International Standardization Organization, pp. 1–34. Khashaba RM, Moussa MM, Chutkan NB, Borke JL (2011) The response of subcutaneous connective tissue to newly developed calcium phosphate-based root canal sealers. International Endodontic Journal 44, 342–52. Lacativa AM, Loyola AM, Sousa CJ (2012) Histological evaluation of bone response to pediatric endodontic pastes: an experimental study in guinea pig. Brazilian Dental Journal 23, 635–44. Marques AA, Sponchiado EC Jr, Garcia LF, Garrido AD, Francßa SC, Lia RC (2011) Morphological analysis of tissue reaction caused by a new endodontic paste in subcutaneous tissue of rats. Journal of Conservative Dentistry 14, 309–13. Mohammadi Z, Dummer PMH (2011) Properties and applications of calcium hydroxide in endodontics and dental traumatology. International Endodontic Journal 44, 697–730. Molander A, Dahlen G (2003) Evaluation of the antibacterial potential of tetracycline or erythromycin mixed with calcium hydroxide as intracanal dressing against Enterococcus faecalis in vivo. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology 96, 744–50. Mutoh N, Tani-Ishii N (2011) A biocompatible model for evaluation of the responses of rat periapical tissue to a new zinc oxide-eugenol sealer. Dental Materials Journal 30, 176–82. Olsson B, Sliwkowski A, Langeland K (1981) Subcutaneous implantation for the biological evaluation of endodontic materials. Journal of Endodontics 7, 355–67. Pereira MSS, Faria G, da Silva LAB, Tanomaru-Filho M, Kuga MC, Rossi MA (2012) Response of mice connective tissue to intracanal dressings containing chlorhexidine. Microscopy Research and Technique 75, 1653–8. Peters OA (2013) Research that matters – biocompatibility and cytotoxicity screening. International Endodontic Journal 46, 195–7.
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Pinky C, Shashibhushan KK, Subbareddy VV (2011) Endodontic treatment of necrosed primary teeth using two different combinations of antibacterial drugs: an in vivo study. Journal of the Indian Society of Pedodontics and Preventive Dentistry 29, 121–7. Putt FA (1972) Manual of Histopathological Staining Methods. New York: John Wiley and Sons. Queiroz AM, Assed S, Consolaro A et al. (2011) Subcutaneous connective tissue response to primary root canal filling materials. Brazilian Dental Journal 22, 203–11. Ranly DM, Garcia-Godoy F (2000) Current and potential pulp therapies for primary and young permanent teeth. Journal of Dentistry 28, 153–61. Semenoff TA, Semenoff Segundo A, de Figueiredo JA (2008) Biocompatibility of different intracanal medications in rat bucal submucosa tissue. Journal of Applied Oral Science 16, 12–7. Serhan CN (2010) Novel lipid mediators and resolution mechanisms in acute inflammation: to resolve or not? American Journal of Pathology 177, 1576–91. Silva RA, Assed S, Nelson-Filho P, Silva LA, Consolaro A (2009) Subcutaneous tissue response of isogenic mice to calcium hydroxide-based pastes with chlorhexidine. Brazilian Dental Journal 20, 99–106. Silva LAB, Leonardo MR, Oliveira DSB et al. (2010) Histopathological evaluation of root canal filling materials for primary teeth. Brazilian Dental Journal 21, 38–45. Sousa CJA, Loyola AM, Versiani MA, Biffi JCG, Oliveira RP, Pascon EA (2004) A comparative histological evaluation
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of the biocompatibility of materials used in apical surgery. International Endodontic Journal 37, 738–48. de Sousa DL, de Sousa RB, Pinto DN, Neto JJ, de Carvalho CB, de Almeida PC (2011) Antibacterial effects of chemomechanical instrumentation and calcium hydroxide in primary teethwith pulp necrosis. Pediatric Dentistry 33, 307–11. Takushige T, Cruz EV, Asgor Moral A, Hoshino E (2004) Endodontic treatment of primary teeth using a combination of antibacterial drugs. International Endodontic Journal 37, 132–8. Torneck CD (1966) Reaction of rat connective tissue to polyethylene tube implants. I. Oral Surgery, Oral Medicine, Oral Pathology 21, 379–87. Trairatvorakul C, Detsomboonrat P (2012) Success rates of a mixture of ciprofloxacin, metronidazole, and minocycline antibiotics used in the non-instrumentation endodontic treatment of mandibular primary molars with carious pulpal involvement. International Journal of Paediatric Dentistry 22, 217–27. Viola NV, Guerreiro-Tanomaru JM, da Silva GF, Sasso-Cerri E, Tanomaru-Filho M, Cerri PS (2012) Biocompatibility of an experimental MTA sealer implanted in the rat subcutaneous: quantitative and immunohistochemical evaluation. Journal of Biomedical Materials Research Part B: Applied Biomaterials 100, 1773–81. Wataha JC (2001) Principles of biocompatibility for dental practitioners. Journal of Prosthetic Dentistry 86, 203–9.
© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
Biocompatibility of root filling pastes used in primary teeth
nior2, M. S. Rizzo3, R. D. Moura4, M. S. Moura5, M. D. M. C. C. B. Lima1, A. M. Conde Ju 5 5 Lima & L. F. A. D. Moura 1
Postgraduate Programme in Dentistry, Federal University of Piauı (UFPI), Teresina; 2Department of Morphology, Federal University of Piauı (UFPI), Teresina; 3Department of Clinic and Veterinary Surgery, Federal University of Piauı (UFPI), ao Paulo (USP), S~ ao Paulo; and 5Department of Pathology Teresina; 4Postgraduate Programme in Pathology, University of S~ and Dentistry Clinic, Federal University of Piauı (UFPI), Teresina, Brazil
Abstract nior AM, Rizzo MS, Moura RD, Lima CCB, Conde Ju Moura MS, Lima MDM, Moura LFAD. Biocompatibility of root filling pastes used in primary teeth. International Endodontic Journal.
Aim To evaluate the biocompatibility of two pastes designed to fill the root canals of primary teeth. Methodology A study group of 54 mice received subcutaneous tissue implants of polyethylene tubes containing CTZ or calcium hydroxide paste or, as a negative control, empty tubes. Biocompatibility was evaluated on days 7, 21 and 63, yielding a total of nine groups of six animals each. Following the experimental intervals, the implant areas were removed and subjected to histologic processing. After the tissues were stained with HE and Masson trichrome, two pathologists performed a histologic analysis of the samples in a blinded manner. Collagen fibre formation, tissue thickness and inflammatory cell infiltration were analysed qualitatively. Quantitative morphometry was performed for the thickness, perimeter length and tissue
Introduction The complex morphology of root canal systems and the root resorption of primary molars are uncontrollable factors that may hinder proper chemical–mechanical
Correspondence: L ucia de F atima Almeida de Deus Moura, Department of Pathology and Dentistry Clinic, Federal University of Piauı, Rua Angelica, 1650, Bairro de Fatima, CEP 64049-532, Teresina, Piauı, Brazil (Tel.: +55 86 3233 3050; e-mail: [email protected]).
© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
area of the region in direct contact with the open tube. with the Tukey post-test and Kruskal–Wallis analysis followed by Dunn’s post-test, with significance established as P < 0.05, were used for data analysis. Results At 7 days, all groups had severe acute inflammatory infiltrates. Inflammation was reduced at 21 days in the CTZ paste group. Mild chronic inflammatory infiltrates were observed after 63 days in the CTZ and Ca(OH)2 paste groups; these groups also showed a significant decrease in collagen fibre density (P < 0.05), which was not observed in the control group. The average tissue thickness, perimeter length and area in contact with the tube decreased during the experimental periods in all groups. Conclusion The CTZ and calcium hydroxide pastes demonstrated biocompatibility with subcutaneous tissue in this experimental model. ANOVA
Keywords: biocompatibility, calcium hydroxide, primary teeth, root canal filling materials, subcutaneous tissue. Received 26 August 2013; accepted 29 May 2014
canal disinfection in primary teeth with pulp necrosis (Harini Priya et al. 2010, de Sousa et al. 2011). These conditions may also impede the development of permanent tooth follicles as well as the establishment of accurate working length (Molander & Dahlen 2003, Barja-Fidalgo et al. 2011, Gondim et al. 2012). Most of the recommended techniques for filling root canals of primary teeth propose the use of instrumentation and the chemo-mechanical preparation of the root canal system (American Academy of Pediatric
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Dentistry 2012). The use of calcium hydroxide (Ca (OH)2) paste predominates over other techniques for the pulp therapy of primary teeth. As a root filling material, Ca(OH)2 paste demonstrates antibacterial action, biocompatibility and tissue repair capacity (Hauman & Love 2003b, Mohammadi & Dummer 2011). Alternative pulp therapies that have been developed include those that recommend restricting treatment to the pulp chamber and methods that may promote the disinfection or sterilization of root canals with necrotic pulps, as in the use of antibiotic-containing pastes (Cappiello 1964, Takushige et al. 2004, Pinky et al. 2011, Trairatvorakul & Detsomboonrat 2012). Cappiello (1964) developed a paste for filling root canals in primary teeth. The paste comprises chloramphenicol, tetracycline and zinc oxide with eugenol as a vehicle and is currently known as CTZ paste (powder base provided by a pharmacy in proportions of 1 : 1 : 2 chloramphenicol/tetracycline/zinc oxide, respectively). This method obviates the need for canal instrumentation, thereby facilitating its use in young children and users of public health services (Cappiello 1964). Successful endodontic treatment of teeth with pulp necrosis requires reducing or eliminating infection within the root canal system. The medicaments used in the pulp therapy of primary teeth should possess antimicrobial activity and be biocompatible, particularly given the proximity of contact in the furcation and periapical regions (Silva et al. 2010, Barja-Fidalgo et al. 2011). Biocompatibility is the ability of a material that is in contact with a tissue to promote an appropriate biological response with minimal inflammatory reactions. Methods that deploy substances in the subcutaneous tissues of laboratory animals are commonly used to evaluate the biocompatibility of endodontic materials (Hauman & Love 2003a, Viola et al. 2012). The purpose of this study was to evaluate the biocompatibility of two pastes for the root canal treatment of primary teeth (CTZ and calcium hydroxide pastes). The null hypothesis was that there is no difference between the biocompatibility of CTZ and calcium hydroxide pastes in mouse subcutaneous tissue.
Materials and methods Ethical approval The procedures used in this study were in accordance with a protocol reviewed and approved by the Ethics Committee on Animal Experimentation, Federal
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University of Piauı (UFPI) (opinion No. 067/12) and with the ethical guidelines for animal experimentation advocated in the ‘European Communities Council Directive’ of 24 November 1986 (86/609/EEC) regarding recommendations for the care and use of laboratory animals. This experimental trial began in November 2012 and was concluded in May 2013 upon euthanasia of the last test animal.
Animal model A total of 54 mice (Mus musculus) were used. The animals were Swiss albino males from 6 to 8 weeks of age that weighed between 15 and 30 g. Throughout the experiment, the animals were housed in a vivarium with standard rations and access to water ad libitum. All animals were examined every 2 days and monitored for local or systemic changes. The animals were wormed with Baskenâ suspension (14.5 mg pyrantel pamoate + 9.5 mg oxantel pamoate kg 1), which was administered orally in a single dose 7 days prior to the onset of the experimental procedural.
Root canal pastes The biocompatibility of the CTZ and calcium hydroxide pastes was tested using an empty tube as a negative control. Histological analysis was performed on the tissue samples for each material at three different intervals: 7, 21 and 63 days. The tissue response was analysed for nine groups with six animals per group, which were distributed as follows: Group I – six animals received a polyethylene tube containing CTZ paste for 7 days; Group II – six animals received a polyethylene tube containing CTZ paste for 21 days; Group III – six animals received a polyethylene tube containing CTZ paste for 63 days; Group IV – six animals received a polyethylene tube containing calcium hydroxide paste for 7 days; Group V – six animals received a polyethylene tube containing calcium hydroxide paste for 21 days; Group VI – six animals received a polyethylene tube containing calcium hydroxide paste for 63 days; Group VII – six animals received an empty polyethylene tube for 7 days; Group VIII – six animals received an empty polyethylene tube for 21 days; and Group IX – six animals received an empty polyethylene tube for 63 days. The micronized powder base for the CTZ paste was provided in 250-mg capsules by a compounding
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pharmacy (Teresina, PI, Brazil), and the paste comprised chloramphenicol 62.5 mg, tetracycline 62.5 mg and zinc oxide 125 mg with 0.1 mL of eugenol vehicle (Biodin^ amica, Ibipor~a, PR, Brazil). Calcium hydroxide (Ca(OH)2) powder (analytical grade) (Biodin^ amica) was provided in 250-mg capsules and reconstituted in 0.2 mL of distilled water (Isofarma, Eusebio, CE, Brazil). To prepare the two pastes, the powders were dispensed onto sterile glass plates and mixed with their respective vehicles by a metal spatula at the time of use.
Experimental design for subcutaneous implantation Animals were pre-medicated with Tramadol at a dose of 2 mg kg 1 (Tramadol hydrochloride 50 mg mL 1, Hipolaborâ; Sabar a, MG, Brazil) by deep intramuscular injection with a 1-mL syringe. After 10 minutes, anaesthesia was induced using a combination of xylazine (10 mg kg 1) (xylazine 2% Xilazin â; Syntec, Cotia, SP, Brazil) and ketamine (80 mg kg 1) (ketamine hydrochloride 50 mg mL 1, Clortaminaâ; BioChimico, Itatiaia, RJ, Brazil) prepared in the same syringe and administered by deep intramuscular injection. The back of the animal was shaved, and the surgical region was disinfected with a 1% solution of chlorhexidine gluconate. In preparation for the dorsal implantation in each animal, polyethylene tubes that were 1 mm in internal diameter (peripheral catheter type scalp No. 27 Solidorâ; Shijiazhuang, China) were sterilized in ethylene oxide and closed at one end with an Allis forceps. The tubes were heated, sliced into 1-cm segments and filled with the material to be tested. After a 0.5-cm linear incision was made at the caudal lumbar dorsum, the soft tissue was dissected, and a tube was inserted into the tissue. The skin incision was closed with 5.0 nylon sutures (Shalonâ Sutures; S~ ao Luis de Montes Belo, GO, Brazil). For the first 24 h following implantation, the animals received tramadol analgesia (2 mg kg 1), which was administered by deep intramuscular injection every 6 h. At the end of 7, 21 or 63 days, animals from each group were pre-medicated with tramadol (2 mg kg 1) by deep intramuscular injection and euthanized with an anaesthetic combination of xylazine (10 mg kg 1) and ketamine (80 mg kg 1) prepared in the same syringe and administered by deep intramuscular injection. The implant area, which comprised polyethylene
© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
tubing, subcutaneous tissue and skin, was removed and fixed in buffered 4% paraformaldehyde for 24 h.
Histologic analysis Following fixation, the polyethylene tubes were removed from the fixative, dehydrated in increasing concentrations of alcohol, cleared in xylene, embedded in paraffin and blocked. The blocks were cut using a microtome (Leica RM 2245) to yield 5-lmthick longitudinal semi-serial sections. Tissues were stained with haematoxylin and eosin (HE) and Masson trichrome (Putt 1972). The sections were examined under a binocular light microscope (Olympus, Tokyo, Japan) at 100, 200 and 4009 magnification and digitally photographed. A histologic analysis was performed by two pathologists, who were blind to the experimental conditions, at different times. Both examiners analysed the same fields. The Masson trichrome and HE stains were used to analyse the collagen fibre formation and thickness, respectively, at 2009 magnification. The inflammatory infiltrate was analysed at 4009 magnification with HE stain. The intra-examiner agreement was based on two histologic evaluations performed at different intervals on 10% of the sections, which were arranged in order from the smallest to the largest increase (j = 0.80 and 0.81). The interexaminer agreement (j = 0.79) indicated interexaminer reliability. The tissues that contacted the open end of the tubes were analysed and graded for the following histologic findings: 1. formation of collagen fibres, 2. tissue thickness and 3. inflammatory infiltrate. The density of collagen fibre formation was graded as 0 (absent), 1 (mild – collagen fibres arranged similarly to normal connective tissue), 2 (moderate – individual collagen fibres were observed, but did not display linear features or a typical wavy microanatomy) and 3 (severe – very dense, individual collagen fibres cannot be distinguished) (Silva et al. 2009, Queiroz et al. 2011). The tissue thickness was assessed by the extent of fibroblast cell layers formed and the presence of macrophages in the peripheral contact region of the tube and classified as follows: 0 (normal – no layer), 1 (slightly increased – 1 to 3 layers), 2 (moderately increased – 4 to 10 layers) and 3 (intensely increased – more than 10 layers). The inflammatory infiltrate was analysed qualitatively to assess the characteristics and concentration of polymorphonuclear cells (PMN) and mononuclear cells, which were classified as
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follows: 0 (no cells), 1 (mild – 1 to 10 cells), 2 (moderate – 11 to 20 inflammatory cells) and 3 (intense – inflammatory cell concentration ≥21 cells) (Silva et al. 2009, Queiroz et al. 2011).
Morphometric analysis Morphometry, via quantitative analysis of the tissue area that directly contacted the open part of the tube, was performed using imaging software (Leica QWin – Image Processing and Analysis Software Leica D1000, version 4.1; Cambridge, UK). Estimated values for the tissue thickness (lm), perimeter (lm) and area (lm2) were obtained by image analysis of the photomicrographs.
Statistical analysis Data were tabulated and analysed using SPSS version 18.0 (SPSS Inc., Chicago, IL, USA). The Shapiro–Wilk test was used to test the null hypothesis. ANOVA with the Tukey post-test was used to verify differences between the areas and perimeter thicknesses, and the data grading scores were analysed by the Kruskal– Wallis test followed by Dunn’s post-test, with significance established as P < 0.05 (Fig. 1).
Results Upon removal, all implants were covered with a visible, palpable and transparent fibrous capsule. There were no signs of rejection in the specimens. The tubes containing CTZ paste displayed progressive darkening over time. The results from the qualitative histologic analysis of the collagen fibre formation, thickness and inflammatory infiltration of connective tissue adjacent to the open ends of the polyethylene tubes are presented in Table 1 and Figs 2, 3 and 4. When the same treatment was compared for different experimental periods (7, 21 and 63 days), significant differences were observed in the reduction of collagen fibre formation (P < 0.05) and inflammatory cell infiltration (P < 0.05) for the specimens that were treated with calcium hydroxide paste. In the group implanted with an empty tube, increased formation of collagen fibres (P < 0.05) was observed, and no significant difference was observed with the CTZ paste at different times (P > 0.05). The mean values with standard deviations for the thickness (lm), perimeter (lm) and area (lm2) of the reactive tissue in direct contact with the open tubes are presented in Table 2.
Figure 1 Study flowchart.
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Moderately increased (Fig. 3a) Mild to moderately increased (Fig. 3d) Severely increased (Fig. 3g)
Mild collagen fibre density (Fig. 2d) Mild collagen fibre density (Fig. 2g)
Severe; mononuclear and occasional neutrophils present (Fig. 4c) Mild; predominantly mononuclear with occasional neutrophils (Fig. 4f) Mild; predominantly mononuclear with occasional neutrophils (Fig. 4i)
Moderate; predominantly mononuclear, with the possible presence of neutrophils (Fig. 4e) Moderate; with macrophages, neutrophils and neovascularization (Fig. 4h)
Moderately increased (Fig. 3c) Mild to moderately increased (Fig. 3f) Slightly increased (Fig. 3i)
Collagen fibres admixed with extracellular matrix without any typical linear or wavy forms; individual collagen fibres could not be distinguished (Fig. 2c) Moderate (Fig. 2f) Mild to moderate (Fig. 2i)
63c
Severe; presence of macrophages, lymphocytes, neutrophils and newly formed vessels (Fig. 4b)
Severely increased (Fig. 3b) Slightly increased (Fig. 3e) Moderately increased, marked presence of fibroblasts (Fig. 3h)
Mild collagen fibre density (Fig. 2e) Individual collagen fibres observed as in normal connective tissue (Fig. 2h)
Individual collagen fibres and alternating extracellular matrix without linear features or wavy fibres (Fig. 2b)
21b
Experimental period (days)
P: Kruskal–Wallis. a There was no statistically significant difference between groups (P > 0.05). b There was no statistically significant difference between groups (P > 0.05). c There was a statistically significant difference between the groups in the collagen fibre formation during this period (P < 0.05).
Inflammatory infiltrate (Fig. 4a–j) Empty tube Severe; presence of lymphocytes, macrophages, neutrophils, and newly formed vessels (Fig. 4a) Calcium hydroxide paste Moderate; mixed macrophages and neutrophils present (Fig. 4d) CTZ paste Severe; predominantly mononuclear, with neutrophils present (Fig. 4g)
Tissue thickness (Fig. 3a–j) Empty tube Calcium hydroxide paste CTZ paste
Calcium hydroxide paste CTZ paste
Formation of collagen fibres (Fig. 2a–j) Empty tube Mild to moderate (Fig. 2a)
Material tested
a
Table 1 Qualitative histologic analysis of the collagen fibre formation, thickness and inflammatory cell infiltration of the connective tissue adjacent to the open ends of the polyethylene tubing
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Figure 2 (a–i) Photomicrographs stained with Masson trichrome showing the formation of collagen fibres in the connective tissue adjacent to the open ends of the polyethylene tubes in contact with the empty tube (a, d, g), calcium hydroxide paste (Ca (OH)2) (b, e, h) and CTZ paste (c, f, i), at 7, 21 and 63 days, respectively (2009 magnification). (j) Groups for the qualitative analysis of the collagen fibre formation. (1) Space occupied by the tube and (2) subcutaneous tissue.
Discussion A great challenge facing Paediatric Dentistry is to develop biomaterials that possess antimicrobial properties, in addition to being biocompatible and resorb-
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able, for primary teeth with necrotic pulps (Silva et al. 2010, Barja-Fidalgo et al. 2011, Queiroz et al. 2011). CTZ paste has great potential as a treatment option for endodontically compromised primary teeth in
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Lima et al. Materials biocompatibility
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Figure 3 (a–i) Photomicrographs showing the HE staining for the tissue thickness adjacent to the open end of polyethylene tube in contact with the empty tube (a, d, g), calcium hydroxide paste (Ca(OH)2) (b, e, h) and CTZ paste (c, f, i), at 7, 21 and 63 days, respectively (2009 magnification). (j) Groups for the qualitative analysis of tissue thickness. (1) Space occupied by the tubing, (2) subcutaneous tissue, (*) neovascularization, (f) fibroblasts, (l) lymphocytes, (m) macrophages and (n) neutrophils.
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Figure 4 (a–i) Photomicrographs of HE-stained tissue showing inflammatory infiltrate in the connective tissue adjacent to the open end of the polyethylene tube in contact with the empty tube (a, d, g), calcium hydroxide paste (Ca(OH)2) (b, e, h) and CTZ paste (c, f, i), at 7, 21 and 63 days, respectively (4009 magnification). (j) Groups for the qualitative analysis of the inflammatory reaction. (1) Space occupied by the tubing, (2) subcutaneous tissue, (*) neovascularization, (f) fibroblasts, (l) lymphocytes, (m) macrophages and (n) neutrophils.
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Table 2 Mean values with standard deviations for thickness (lm), perimeter (lm) and area (lm2) of tissue reaction
Thickness (lm) CTZ Ca(OH)2 Control P Perimeter (lm) CTZ Ca(OH)2 Control P Area (lm2) CTZ Ca(OH)2 Control P
Day 7 M (SD)
Day 21 M (SD)
Day 63 M (SD)
135.9 (59.0) 84.7 (17.2) 108.3 (41.6) 0.091
121.4 (69.6) 92.0 (28.5) 87.1 (22.6) 0.389
88.8 (41.0) 76.4 (26.1) 70.4 (31.8) 0.634
0.376 0.552 0.121
1046.1 (252.3) 884.4 (253.8) 1079.6 (73.1) 0.261
950.0 (211.8) 894.3 (143.0) 880.4 (115.2) 0.738
941.2 (179.0) 916.7 (173.4) 763.8 (190.9) 0.218
0.656 0.958 0.004
53554.9 (16442.2) 32132.6 (11382.7) 49362.2 (9999.3) 0.027
47344.8 (27953.0) 32404.8 (9832.4) 32561.9 (12713.6) 0.308
33816.3 (13998.1) 24996.0 (1335.6) 23483.9 (13930.3) 0.396
0.261 0.473 0.008
P*
M = mean; SD = standard deviation. P: ANOVA test, applied between the materials in same experimental period. * P: ANOVA test, applied for each material between the different experimental periods.
young children with behavioural problems. Another application for CTZ paste is for users of public health services because of the large patient loads in such facilities and the ease with which general practitioners can perform these procedures due to the technical simplicity, excellent clinical outcomes and cost-effectiveness (Cappiello 1964, Bruno et al. 2007). There is a lack of scientific literature on the use of CTZ paste in clinical settings. This study is part of a macro-development project that encompasses research on the performance and antimicrobial action of CTZ paste and its components. Biocompatibility is the ability of an applied material or substance to trigger the intended response in the host. That is, although a biocompatible material may not be inert, the reaction caused by a test material must not pose an unacceptable physiological hazard when compared with other scientifically approved materials (Wataha 2001, Peters 2013). Three tests are recommended for evaluating the biocompatibility of endodontic materials: initial, secondary and application. The method used in this study was a sub-type that is used to evaluate biological tissue responses around the test material and to compare such results to biocompatible controls with established scientific acceptability (Olsson et al. 1981, ISO 7405 1996, ISO 10993-6 2007a, ISO 10993-12 2007b, Hauman & Love 2003a, Silva et al. 2009, Pereira et al. 2012). Subcutaneous implants, which were introduced by Torneck (1966), are used most commonly in dentistry.
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Compatibility with living tissue in the periapical or interradicular region is one of the most important properties for materials used in pulp therapy of primary teeth due to the occurrence of root resorption and the presence of anatomical variations that may jeopardize periodontal and periapical tissues, which may in turn endanger the development of permanent teeth (Lacativa et al. 2012). Polyethylene tube implants are used experimentally to simulate the relationship between the tooth apex and periapical tissues because the material within the tubes diffuses in a restricted area (Olsson et al. 1981, International Organization for Standardization 2007a, Silva et al. 2009, Khashaba et al. 2011, Marques et al. 2011, Mutoh & Tani-Ishii 2011). In this study, the groups in which empty polyethylene tubes were implanted had higher scores in the qualitative histologic analysis for fibroplasia and had significant differences in the perimeter and area of the tissue regions in direct contact with the open tube. This result was expected because no materials were introduced that could induce an acute inflammatory response or reaction. However, other studies have reported responses to empty tubes, albeit less intense than those observed for the experimental parameters analysed at the time (Silva et al. 2009, Queiroz et al. 2011). Significant reductions were observed in the density of collagen fibres and the inflammatory reaction when testing the calcium hydroxide paste for different experimental periods. Calcium hydroxide is widely
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used in dentistry due to its biocompatible properties and antimicrobial, anti-inflammatory and reparative activities (Hauman & Love 2003a,b, Mohammadi & Dummer 2011, de Sousa et al. 2011, Gondim et al. 2012). The biocompatibility results obtained in this study for calcium hydroxide paste are consistent with those reported by several authors (Holland et al. 2001, Semenoff et al. 2008, Silva et al. 2010, Andolfatto et al. 2012). In the present study, the tubes containing CTZ paste displayed progressive darkening over time, which may have been caused by the tetracycline component. The CTZ paste was associated with an initial intense inflammatory response during the first days of the experiment. At the end of the experiment, a mild chronic inflammatory process was observed, with a qualitative and quantitative decrease in the density of collagen fibres and tissue thickness. This finding suggests that CTZ was biocompatible and showed therapeutic properties. In a previous study, histopathologic analysis of pulpotomy performed in dogs’ teeth treated with CTZ paste demonstrated the occurrence of an initial intense inflammatory process in the coronal pulp with reduced inflammation at the end of the experiment (Bruno et al. 2007). However, the literature lacks studies on the biocompatibility of CTZ paste; thus, it was not possible to compare the results obtained in this study with those of other published studies. The normal reaction to any tissue injury tends to manifest as an acute neutrophilic inflammatory infiltrate (Serhan 2010). Given that acute histopathologic tissue behaviour is similar across all groups, studies that aim to test the biocompatibility of a drug that may cause a tissue reaction should be focused on chronic inflammatory reactions. In this study, the pastes and the control group showed variable chronic inflammatory processes at different experimental times. A histologic hallmark of successful pulp therapy is the development of reparative tissue (Torneck 1966). In the present study, a significant difference in the density of collagen fibres was observed between the two pastes at the end of the experiment. The difference was characterized by a decrease in the collagen fibres in specimens containing either the CTZ or the calcium hydroxide paste compared with the negative control. This finding is important because the two pastes behaved in manner that favours tissue repair. The results are consistent with other studies that reported similar methodologies (Sousa et al. 2004, Batista et al.
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2007, Silva et al. 2010, Marques et al. 2011, GomesFilho et al. 2012). Other materials are used as pastes for the root canal treatment of primary teeth, including pastes containing zinc oxide and eugenol (ZOE) with iodoform or a paste that combines iodoform and calcium hydroxide (Ranly & Garcia-Godoy 2000, American Academy of Pediatric Dentistry 2012). Studies with ZOE as a root canal filling material reported a chronic inflammatory reaction and slow resorption in relation to the tooth (Fuks 2000, Hauman & Love 2003b). The iodoform pastes, when combined with calcium hydroxide, exhibit good biological response and antimicrobial properties and are more easily reabsorbed at the periapical region (Ranly & Garcia-Godoy 2000). However, the techniques proposed for using these materials recommend the use of instrumentation and chemical–mechanical systems for the root canals of primary teeth (American Academy of Pediatric Dentistry 2012). The limitations of this study are inherent to preclinical experimental studies. Although the results presented here do not reflect a complete analysis of the reactions that occur in the pulp, periapical or interradicular regions, the results are important for the preliminary assessment of the irritative potential of the tested materials and subsequent biological responses.
Conclusion The initial inflammatory reactions in mice induced by CTZ and calcium hydroxide pastes regressed during the experimental periods; in addition, these pastes resulted in less-dense fibrous connective tissue, compared with the results using the empty control. These reactions support the biocompatibility of the two pastes.
Acknowledgements This study was supported financial by a scholarship grant from the Coordination of Improvement of Higher Education Personnel (CAPES).
Author contributions CCBL participated in the project design and data collection and analysis and interpretation of data and wrote the article. AMCJ participated in the project design and data collection and analysis and interpretation of data and wrote the article. MSR
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participated in the analysis and interpretation of data and wrote the article. RDM participated in the analysis of data. MSM revised the article. MDML participated in the study preparation and revised the article. LFADM conceived the study, participated in the study preparation and coordination, and wrote the article. All of the authors read and approved the final manuscript.
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