- Email: [email protected]
In vivo evaluation of the biocompatibility of a new resin-based obturation system
In vivo evaluation of the biocompatibility of a new resin-based obturation system Emel Olga Onay, DDS, PhD,a Mete Ungor, DDS, PhD,b and Binnaz Handan Ozdemir, MD, PhD,c Ankara, Turkey BASKENT UNIVERSITY
Objective. The aim of this study was to evaluate the in vivo biocompatibility of the new resin-based Epiphany-Resilon root canal filling system after implantation in rat connective tissue. Study design. Thirty-six female Wistar albino rats, 9 months old, weighing 200 to 220 g, were used to evaluate the biocompatibility. Four subcutaneous pockets were created in the back of the rats, and each material (Resilon, guttapercha, a Teflon tube containing Epiphany, and an empty Teflon tube) was implanted into a specific dorsal site. The empty Teflon tubes were used as control. After 1, 4, and 8 weeks, the implants were removed with the surrounding tissues. The inflammatory tissue reactions were graded after a histopathologic examination. Results. At each time period, there was practically no difference in the reaction of all the implant materials (P ⬎ .05). The intensity of the reaction had diminished by the 4-week observation period, and this reduction continued through the 8-week observation period. Conclusion. All the tested materials showed an acceptable biocompatibility. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;104:e60-e66)
The three-dimensional hermetic filling of the root canal system after suitable cleaning, shaping, and disinfection is one of the goals to be reached in the achievement of a successful endodontic treatment.1,2 The endodontic filling is generally composed of a combination of core materials, such as gutta-percha and a sealer. Because these materials will be in direct contact with periapical tissues for a prolonged period of time, their biocompatibility is of primary importance.3 Among the wide spectrum of root canal filling materials, a new methacrylate resin-based endodontic sealer (Epiphany) and a polycaprolactone-containing root canal filling material (Resilon) were recently introduced into the market. The Epiphany sealer contains bisphenol-A diglycidyl dimethacrylate (BisGMA), ethoxylated BisGMA, urethane dimethacrylate, hydrophilic difunctional methacrylates, silane-treated barium borosilicate glasses, barium sulfate, silica, calcium hydroxide, bismuth oxychloride with amines, peroxide, photo initiator, stabilizers, and pigment.4 Resilon is a a
Research Fellow, Department of Endodontics, Faculty of Dentistry, Baskent University. b Professor, Department of Endodontics, Faculty of Dentistry, Baskent University. c Associate Professor, Department of Pathology, Faculty of Medicine, Baskent University. Received for publication Aug 30, 2006; returned for revision Feb 23, 2007; accepted for publication Mar 2, 2007. 1079-2104/$ - see front matter © 2007 Mosby, Inc. All rights reserved. doi:10.1016/j.tripleo.2007.03.006
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thermoplastic synthetic resin material that is based on polymers of polyester and contains a bifunctional methacrylate resin, bioactive glass, and radiopaque fillers. It performs like gutta-percha, has the same handling properties, and for retreatment purposes may be softened with heat or dissolved with solvents like chloroform.5 This product is used in combination with a self-etching primer to create a solid monoblock. The primer is an aqueous solution of an acidic monomer. The purpose of the present study was to analyze the biocompatibility of Epiphany and Resilon when implanted into the subcutaneous connective tissue of the rat. MATERIAL AND METHODS Thirty-six female Wistar albino rats, 9 months old, weighing 200 to 220 g were used in this study. Approval for the animal use protocol presented below was sought and given by the Animal Ethic Committees at Baskent University. After ether inhalation, the animals were anesthetized by the administration of ketamine HCl (50 mg/kg) and 7 mg/kg xylazine, intraperitoneally. The back of the animal was shaved and disinfected with 5% iodine in ethanol. Four separate pockets were created by blunt dissection to a depth of 20 mm to implant the materials in the subcutaneous tissue. A Teflon tube (Mediflon; Eastern Medikit LTD., Delhi, India) containing freshly mixed Epiphany sealer (Pentron Clinical Technologies, LLC, Wallingford, CT) according the manufacturer’s instructions, a 10-mm-long. 04 taper Resilon point (Pentron),
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and a 10-mm-long .04 taper gutta-percha point (Diadent Group International Inc., Chongju, Korea) were then placed into each pocket. Empty Teflon tubes, 10 mm in length with an inner diameter of 1.3 mm, were used as the control. Finally, the incisions were closed with surgical gut sutures. At the end of 1, 4, and 8 weeks, the animals were premedicated with ketamine HCl (50 mg/kg) and xylazine (7 mg/kg) and then killed with an overdose of thiopental sodium (150 mg/kg). Implant materials were removed with the surrounding tissue and immersed in 10% buffered formalin. After fixing the tissue for 48 hours, it was processed for paraffin embedding. Paraffin blocks were cut in serial sections (5 slides per specimen) with the microtome set at 4 m. Sections were mounted on glass slides and then stained with hematoxylin and eosin. Selected slides were stained with Masson trichrome for identification of collagen. Tissue reactions such as inflammatory response, the infiltration of mast cells, fibroblast proliferation, and vascular changes were evaluated and recorded. The criteria for scoring the stromal inflammatory response are as follows: ● ●
●
●
grade 0 (no reaction): no stromal lymphocyte, plasma, and neutrophil leukocyte infiltration grade 1 (mild reaction): stromal lymphocyte, plasma, or neutrophil leukocyte infiltration comprising ⬍20% of all biopsies grade 2 (moderate reaction): stromal lymphocyte, plasma, or neutrophil leukocyte infiltration comprising 20% to 40% of all biopsies grade 3 (severe reaction): stromal lymphocyte, plasma, or neutrophil leukocyte infiltration comprising ⬎40% of all biopsies
The criteria for scoring the infiltration of mast cells are as follows: ● ● ● ●
grade 0 (no reaction): no mast cell infiltration grade 1 (mild reaction): mast cell infiltration comprising ⬍10% of all biopsies grade 2 (moderate reaction): mast cell infiltration comprising 10% to 30% of all biopsies grade 3 (severe reaction): mast cell infiltration comprising ⬎30% of all biopsies
The criteria for scoring the proliferation of fibroblasts are as follows: ● ● ●
grade 0 (no reaction): no fibroblast proliferation, normal collagen fiber morphology grade 1 (mild reaction): mild fibroblast proliferation and mild collagen fiber irregularity grade 2 (moderate reaction): moderate fibroblast proliferation and moderate collagen fiber irregularity.
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Table I. Number of implants and intensity of inflammatory response at different periods of the study Experimental period 1 wk
4 wk
8 wk
Material Teflon Epiphany Gutta-percha Resilon Teflon Epiphany Gutta-percha Resilon Teflon Epiphany Gutta-percha Resilon
No. of Grade Grade Grade Grade implants 0* 1† 2‡ 3§ 12 12 12 12 12 12 12 12 12 12 11 12
— 1 1 — 1 1 2 — 4 2 2 3
4 2 — 3 8 7 8 10 8 9 9 9
4 3 3 3 3 4 2 2 — 1 — —
4 6 8 6 — — — — — — — —
*No reaction. †Mild reaction. ‡Moderate reaction. §Severe reaction.
●
grade 3 (severe reaction): severe fibroblast proliferation and collagen fiber homogenization
The criteria for scoring the vascular changes are as follows: ● ● ●
●
grade 0 (no reaction): no significant vascular proliferation grade 1 (mild reaction): the number of vascular structures in 1 high (40⫻) power field is ⬍25 grade 2 (moderate reaction): the number of vascular structures in 1 high (40⫻) power field is between 25 and 50 grade 3 (severe reaction): the number of vascular structures in 1 high (40⫻) power field is ⬎50
The presence of granulation tissue and foreign body giant cells were also described based on subjective single-blind evaluation and scored as present (1) or absent (0). Statistical analysis was performed using Friedman and Cochran tests for intragroup comparison (1, 4, and 8 weeks) and the Kruskal-Wallis test for intergroup comparison (1, 4, and 8 weeks). RESULTS Macroscopic examination showed that wound healing was satisfactory in all animals. The severity of inflammatory tissue reaction to the implanted materials is presented in Table I. Because of problems inherent in laboratory processing, one of the remaining specimens of guttapercha that was in the 8-week group had to be excluded. One-week observations for group 1 In this group, the intensity of inflammatory reaction was similar among the implant materials, and
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Fig. 1. Epiphany implant at the 1-week observation period. Edema, moderate vascular proliferation (blue arrows), and mild inflammatory infiltrate (red arrow) can be seen alongside the Teflon tube (hematoxylin-eosin, original magnification ⫻200).
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Fig. 3. Resilon implant at the 1-week observation period. Hyalinization (H) and fibroblast proliferation (F) can be seen around the material (hematoxylin-eosin, original magnification ⫻100).
Fig. 2. Gutta-percha implant at the 1-week observation period. Tissue next to material shows disintegration of tissue with inflammatory infiltration (red arrows) and vascular (blue arrows) and fibroblast proliferation (F; hematoxylin-eosin, original magnification ⫻200).
intragroup comparison revealed no statistical differences (P ⬎ .05). A peripheral reaction was in progress, with a moderate to severe inflammatory infiltration noted and exudation in the tissue. Edema, moderate vascular proliferation, and mild inflammatory infiltration were present along the Epiphany implant (Fig. 1). The areas in contact with guttapercha and Resilon showed a severe granulomatous reaction with inflammatory infiltration, vascular and fibroblast proliferation, and also hyalinization (Figs. 2 and 3). Granulation tissue was observed along the side of the empty Teflon tube.
Fig. 4. Epiphany implant at the 4-week observation period. Note the diminishing number of inflammatory cells and fibrosis (hematoxylin-eosin, original magnification ⫻100).
Four-week observations for group 2 The intensity of the tissue responses decreased in all implant materials and was scored as mild to moderate (Figs. 4 and 5). Intragroup comparison revealed no
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Fig. 5. Resilon implant at the 4-week observation period. The intensity of the reaction has diminished (hematoxylin-eosin, original magnification ⫻100).
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Fig. 7. Epiphany implant at the 8-week observation period. Slight inflammation (red arrows) with vascular proliferation can be seen (blue arrows; hematoxylin-eosin, original magnification ⫻200).
Fig. 8. Resilon implant at the 8-week observation period. The tissue located in immediate contact with the implant material is free of inflammation (hematoxylin-eosin, original magnification ⫻100).
Fig. 6. Gutta-percha implant at the 8-week observation period. The intensity of the reaction has diminished; however, there is fibrosis (F) and inflammation (red arrow) (hematoxylin-eosin, original magnification ⫻100).
statistical differences between the materials (P ⬎ .05). Some of these specimens exhibited a thin fibrous capsule, free of inflammatory cells. On the other hand, a severe granulomatous reaction with inflammatory infil-
OOOOE September 2007 0.05 0.05 0.05 0.05 ⬎ ⬎ ⬎ ⬎ P P P P .001 .05 .001 .05 ⬍ ⬍ ⬍ ⬍ P P P P .001 .05 .001 .05 ⬍ ⬍ ⬍ ⬍ P P P P 0.05 0.05 0.05 0.05 ⬎ ⬎ ⬎ ⬎
levels of significance were set at P ⬍ .05. *All
⬍ ⬍ ⬍ ⬍ Epiphany Resilon Gutta-percha Teflon
P P P P
.05 .01 .001 .05
P P P P ⬍ ⬍ ⬍ ⬍
.01 .001 .001 .001
P P P P
⬎ ⬎ ⬎ ⬎
0.05 0.05 0.05 0.05
P P P P
⬎ ⬎ ⬎ ⬎
0.05 0.05 0.05 0.05
P P P P
⬍ ⬍ ⬍ ⬍
.001 .001 .001 .001
P P P P
⬍ ⬍ ⬍ ⬍
.001 .001 .01 .01
P P P P
⬍ ⬍ ⬍ ⬍
.05 .001 .001 .05
P P P P
⬍ ⬍ ⬍ ⬍
.001 .001 .001 .001
P P P P
⬍ ⬍ ⬍ ⬍
.05 .05 .05 .05
P P P P
⬍ ⬍ ⬍ ⬍
.01 .01 .001 .05
P P P P
⬍ ⬍ ⬍ ⬍
.001 .001 .001 .001
P P P P
⬍ ⬍ ⬍ ⬍
.05 .01 .05 .05
P P P P
⬍ ⬍ ⬍ ⬍
.01 .01 .001 .05
P P P P
⬍ ⬍ ⬍ ⬍
.01 .01 .001 .05
P P P P
4-8 wk 1-8 wk 1-4 wk
Presence of foreign body giant cells
4-8 wk 1-8 wk
Fibroblast proliferation
1-4 wk 4-8 wk 1-8 wk
Vascular proliferation
1-4 wk 4-8 wk 1-8 wk
Mast-cell infiltration
1-4 wk 4-8 wk 1-8 wk 1-4 wk
Table II. Intergroup comparison of the periods of the study*
DISCUSSION This study has demonstrated that all the materials implanted into the dorsal connective tissue of rats promoted a moderate to severe inflammatory reaction at the 1-week period, which decreased with time. Connective tissue healing was remarkable for all experimental materials despite the moderate inflammatory reaction observed in only 1 Epiphany sample at the 8-week observation period. The initially moderate to severe inflammatory reaction took place for all experimental and control materials was probably the result of the surgical trauma.6,7 The subcutaneous implantation is considered as a suitable secondary test for evaluation of biocompatibility properties of restorative and endodontic materials. Multiple studies have evaluated sealer biocompatibility by using different implantation vehicles, such as polyethylene tubes,7,8 silicon tubes,6,9 dentin tubes,10 and Teflon tubes.11,12 In this study, Teflon tubes were used because of their inert nature and suitability for bringing a test material into contact with living tissue in a controlled and effective manner.13 Various factors determine the biocompatibility of a resin-based material, particularly the amount and nature of leachable components. Conventional composite resins contain a polymerizable organic matrix that consist of several comonomers (e.g., Bis-GMA, UDMA, EGDMA, TEGDMA, etc.) and various additives that function as coinitiators, stabilizers, or inhibitors.14 Costa et al.7 has shown a moderate to intense inflammatory reaction in many samples of 2 current adhesive resins when implanted into dorsal connective tissue of rats, even at 30 days. The irritative effect of these
1-8 wk
Presence of granulation tissue
Eight-week observations for group 3 The inflammatory reaction varied from none to slight in this period, and intragroup comparison revealed no statistical differences between the materials (P ⬎ .05). The total picture was of very slight inflammation and healing (Figs. 6, 7, and 8). It is noteworthy that neither foreign body giant cells nor granulation tissue were observed at this time period. All the implant materials appeared to be equally well tolerated. The intergroup comparison using the Kruskal-Wallis test with different parameters is summarized in Table II. The intergroup comparison reveals that statistically significant differences exist between the periods of the study, which presented a decrement in the intensity of the tissue responses with time.
1-4 wk
tration and vascular and fibroblast proliferation were seen in the tissues surrounding some of the gutta-percha and Resilon specimens. No foreign body giant cells were observed at this time period.
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Onay et al.
Stromal inflammatory cell infiltration
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materials was attributed to the unreacted monomers that remain after polymerization and induce a persistent local inflammatory reaction. Epiphany root canal sealer also contains a small percentage of BisGMA, ethoxylated BisGMA, urethane dimethacrylate, and hydrophilic difunctional methacrylates. The mild to severe inflammatory reaction at the 1-week period can possibly be attributed to these monomers, which could be displaced to connective tissue. Zmener6 reported on the implantation of a methacrylate-based endodontic sealer, so-called EndoRez (Ultradent Products, Inc., South Jordan, UT), into the connective tissues of rats. EndoRez and Epiphany sealers have similar formulation— urethane dimethacrylate and barium sulfate are common ingredients. The author reported connective tissue healing with time and also reported persistent inflammatory reactions in 2 of the EndoRez specimens, in contact with zinc and barium, from the 120-day observation period. This finding suggests the need for the development of further research with electron microprobe analysis to determine whether an occasional persistent breakdown of the sealer really disturb the periapical healing process. The intraosseous implant technique was used in guinea pigs by Sousa et al.,15 who reported that the Epiphany sealer was the only material that presented intraosseous biocompatibility among the EndoRez and AH Plus sealers. They also observed biocompatibility to Epiphany with bone formation and none to slight inflammatory reaction. These findings are in agreement with our study. Gutta-percha and Resilon can be assumed to be inert because these materials are stable core materials. In addition, the biocompatibility of these materials was to be expected. When the results for the Epiphany sealer were compared with gutta-percha and Resilon, the intensity of inflammatory reaction was similar, and intragroup comparison revealed no statistical differences for all the periods of the study. Merdad et al.16 assessed the cytotoxicity of the components of the Epiphany and Resilon obturation system, compared with the AH Plus sealer and guttapercha, by using the Millipore filter test. The authors reported that both set sealers (24 hours, 48 hours), gutta-percha and Resilon, were characterized by noncytotoxic responses. However, it has also been suggested that Resilon has better biocompatibility than .02 taper gutta-percha (Premier Dental Products Co., Plymouth Meeting, PA), .06 taper gutta-percha (DiaDent), and .06 taper Activ GP (Brasseler USA Dental Instrumentation, Savannah, GA) in an in vitro cell culture system by MTT assay (ATCC; American Type Culture Collection).17 According to the present
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study, it is shown that both the .04 taper gutta-percha and Resilon materials that also standardized in length had similar biocompatibility. Gutta-percha is the main filling material used in root canal treatment. The primary ingredient of a gutta-percha cone is zinc oxide (60%-70%), which provides a major part of the radiopacity. Gutta-percha accounts for approximately 20%, and the remaining ingredients are binders, opaquers, and coloring agents.18 Gutta-percha is considered to have acceptable biocompatibility with the formation of collagenous capsule around the implants, with very little or no inflammatory host response.19,20 In the present study, gutta-percha cones proved to have well-tolerated biocompatibility. The results of the present study demonstrate that all the implanted materials are well tolerated by tissues and have acceptable biocompatibility. However, before extrapolation of these results to the clinical situation in human beings, further studies are necessary to evaluate the suitability of the Epiphany sealer and Resilon points as an endodontic obturation system. REFERENCES 1. Nguyen TN. Obturation of the root canal system. In: Cohen S, Burns RC, editors. Pathways of the pulp. 3rd ed. St. Louis: CV Mosby; 1984. p. 205-15. 2. Schilder H. Filling the root canal in three dimensions. Dent Clin North Am 1967;11:723-44. 3. Orstavik D. Endodontic materials. Adv Dent Res 1988;2:12-24. 4. Teixeira FB, Teixeira ECN, Thompson JY, Trope M. Fracture resistance of roots endodontically treated with a new resin filling material. J Am Dent Assoc 2004;135:646-52. 5. Shipper G, Orstavik D, Teixeira FB, Trope M. An evaluation of microbial leakage in roots filled with a thermoplastic synthetic polymer-based root canal filling material (Resilon). J Endod 2004;30:342-7. 6. Zmener O. Tissue response to a new methacrylate-based root canal sealer: preliminary observations in the subcutaneous connective tissue of rats. J Endod 2004;30:348-51. 7. Costa CA, Teixeira HM, do Nascimento AB, Hebling J. Biocompatibility of two current adhesive resins. J Endod 2000;26:512-6. 8. Molloy D, Goldman M, White RR, Kabani S. Comparative tissue tolerance of a new endodontic sealer. Oral Surg Oral Med Oral Pathol 1992;73:490-3. 9. Kaplan AE, Ormaechea MF, Picca M, Canzobre MC, Ubios AM. Rheological properties and biocompatibility of endodontic sealers. Int Endod J 2003;36:527-32. 10. Holland R, de Souza V, Nery MJ, Bernabe PFE, Filho JAO, Dezan E Jr, et al. Calcium salt deposition in rat connective tissue after the implantation of calcium hydroxide-containing sealers. J Endod 2002;28:173-6. 11. Olsson B, Sliwkowski A, Langeland K. Subcutaneous implantation for the biological evaluation of endodontic material. J Endod 1981;7:355-69. 12. Kim JS, Baek SH, Bae KS. In vivo study on the biocompatibility of newly developed calcium phosphate-based root canal sealers J Endod 2004;30:708-11.
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13. Tronstad L, Barnett F, Flax M. Solubility and biocompatibility of calcium-hydroxide containing root canal sealers. Endod Dent Traumatol 1988;4:153-9. 14. Geurtsen W. Biocompatibility of resin-modified filling materials. Crit Rev Oral Biol Med 2000;11:333-55. 15. Sousa CJ, Montes CR, Pascon EA, Loyola AM, Versiani MA. Comparison of the intraosseous biocompatibility of AH Plus, EndoRez and Epiphany root canal sealers. J Endod 2006;32:656-62. 16. Merdad K, Pascon E, Kulkarni G, Santerre P, Friedman S. Cytotoxicity of Epiphany and Resilon™ assessed by the millipore filter test [abstract]. J Endod 2006;32:247. 17. Zhu Q, Jiang J, Safavi K, Spangberg L. Biocompatibility of Activ GP and Resilon™ cones on L929 cells in vitro [abstract]. J Endod 2006;32:248. 18. Spangberg L. Endodontic treatment of teeth without apical peri-
odontitis. In: Orstavik D, Pitt Ford TR, editors. Essential endodontology. 2nd ed. Cambridge, UK: Blackwell Science; 1999. p. 228. 19. Spangberg L. Biological effects of root-canal-filling materials. Part 7. Reaction of bony tissue to implanted root-canal-filling material in guinea pigs. Odontol Tidskr 1969;77:133-59. 20. Wolfson EM, Seltzer S. Reaction of rat connective tissue to some gutta-percha formulations. J Endod 1975;1:395-402. Reprint requests: Emel Olga Onay, DDS, PhD Faculty of Dentistry Baskent University 11. sokak No: 26 06490 Bahcelievler Ankara, Turkey [email protected]
Objective. The aim of this study was to evaluate the in vivo biocompatibility of the new resin-based Epiphany-Resilon root canal filling system after implantation in rat connective tissue. Study design. Thirty-six female Wistar albino rats, 9 months old, weighing 200 to 220 g, were used to evaluate the biocompatibility. Four subcutaneous pockets were created in the back of the rats, and each material (Resilon, guttapercha, a Teflon tube containing Epiphany, and an empty Teflon tube) was implanted into a specific dorsal site. The empty Teflon tubes were used as control. After 1, 4, and 8 weeks, the implants were removed with the surrounding tissues. The inflammatory tissue reactions were graded after a histopathologic examination. Results. At each time period, there was practically no difference in the reaction of all the implant materials (P ⬎ .05). The intensity of the reaction had diminished by the 4-week observation period, and this reduction continued through the 8-week observation period. Conclusion. All the tested materials showed an acceptable biocompatibility. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;104:e60-e66)
The three-dimensional hermetic filling of the root canal system after suitable cleaning, shaping, and disinfection is one of the goals to be reached in the achievement of a successful endodontic treatment.1,2 The endodontic filling is generally composed of a combination of core materials, such as gutta-percha and a sealer. Because these materials will be in direct contact with periapical tissues for a prolonged period of time, their biocompatibility is of primary importance.3 Among the wide spectrum of root canal filling materials, a new methacrylate resin-based endodontic sealer (Epiphany) and a polycaprolactone-containing root canal filling material (Resilon) were recently introduced into the market. The Epiphany sealer contains bisphenol-A diglycidyl dimethacrylate (BisGMA), ethoxylated BisGMA, urethane dimethacrylate, hydrophilic difunctional methacrylates, silane-treated barium borosilicate glasses, barium sulfate, silica, calcium hydroxide, bismuth oxychloride with amines, peroxide, photo initiator, stabilizers, and pigment.4 Resilon is a a
Research Fellow, Department of Endodontics, Faculty of Dentistry, Baskent University. b Professor, Department of Endodontics, Faculty of Dentistry, Baskent University. c Associate Professor, Department of Pathology, Faculty of Medicine, Baskent University. Received for publication Aug 30, 2006; returned for revision Feb 23, 2007; accepted for publication Mar 2, 2007. 1079-2104/$ - see front matter © 2007 Mosby, Inc. All rights reserved. doi:10.1016/j.tripleo.2007.03.006
e60
thermoplastic synthetic resin material that is based on polymers of polyester and contains a bifunctional methacrylate resin, bioactive glass, and radiopaque fillers. It performs like gutta-percha, has the same handling properties, and for retreatment purposes may be softened with heat or dissolved with solvents like chloroform.5 This product is used in combination with a self-etching primer to create a solid monoblock. The primer is an aqueous solution of an acidic monomer. The purpose of the present study was to analyze the biocompatibility of Epiphany and Resilon when implanted into the subcutaneous connective tissue of the rat. MATERIAL AND METHODS Thirty-six female Wistar albino rats, 9 months old, weighing 200 to 220 g were used in this study. Approval for the animal use protocol presented below was sought and given by the Animal Ethic Committees at Baskent University. After ether inhalation, the animals were anesthetized by the administration of ketamine HCl (50 mg/kg) and 7 mg/kg xylazine, intraperitoneally. The back of the animal was shaved and disinfected with 5% iodine in ethanol. Four separate pockets were created by blunt dissection to a depth of 20 mm to implant the materials in the subcutaneous tissue. A Teflon tube (Mediflon; Eastern Medikit LTD., Delhi, India) containing freshly mixed Epiphany sealer (Pentron Clinical Technologies, LLC, Wallingford, CT) according the manufacturer’s instructions, a 10-mm-long. 04 taper Resilon point (Pentron),
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and a 10-mm-long .04 taper gutta-percha point (Diadent Group International Inc., Chongju, Korea) were then placed into each pocket. Empty Teflon tubes, 10 mm in length with an inner diameter of 1.3 mm, were used as the control. Finally, the incisions were closed with surgical gut sutures. At the end of 1, 4, and 8 weeks, the animals were premedicated with ketamine HCl (50 mg/kg) and xylazine (7 mg/kg) and then killed with an overdose of thiopental sodium (150 mg/kg). Implant materials were removed with the surrounding tissue and immersed in 10% buffered formalin. After fixing the tissue for 48 hours, it was processed for paraffin embedding. Paraffin blocks were cut in serial sections (5 slides per specimen) with the microtome set at 4 m. Sections were mounted on glass slides and then stained with hematoxylin and eosin. Selected slides were stained with Masson trichrome for identification of collagen. Tissue reactions such as inflammatory response, the infiltration of mast cells, fibroblast proliferation, and vascular changes were evaluated and recorded. The criteria for scoring the stromal inflammatory response are as follows: ● ●
●
●
grade 0 (no reaction): no stromal lymphocyte, plasma, and neutrophil leukocyte infiltration grade 1 (mild reaction): stromal lymphocyte, plasma, or neutrophil leukocyte infiltration comprising ⬍20% of all biopsies grade 2 (moderate reaction): stromal lymphocyte, plasma, or neutrophil leukocyte infiltration comprising 20% to 40% of all biopsies grade 3 (severe reaction): stromal lymphocyte, plasma, or neutrophil leukocyte infiltration comprising ⬎40% of all biopsies
The criteria for scoring the infiltration of mast cells are as follows: ● ● ● ●
grade 0 (no reaction): no mast cell infiltration grade 1 (mild reaction): mast cell infiltration comprising ⬍10% of all biopsies grade 2 (moderate reaction): mast cell infiltration comprising 10% to 30% of all biopsies grade 3 (severe reaction): mast cell infiltration comprising ⬎30% of all biopsies
The criteria for scoring the proliferation of fibroblasts are as follows: ● ● ●
grade 0 (no reaction): no fibroblast proliferation, normal collagen fiber morphology grade 1 (mild reaction): mild fibroblast proliferation and mild collagen fiber irregularity grade 2 (moderate reaction): moderate fibroblast proliferation and moderate collagen fiber irregularity.
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Table I. Number of implants and intensity of inflammatory response at different periods of the study Experimental period 1 wk
4 wk
8 wk
Material Teflon Epiphany Gutta-percha Resilon Teflon Epiphany Gutta-percha Resilon Teflon Epiphany Gutta-percha Resilon
No. of Grade Grade Grade Grade implants 0* 1† 2‡ 3§ 12 12 12 12 12 12 12 12 12 12 11 12
— 1 1 — 1 1 2 — 4 2 2 3
4 2 — 3 8 7 8 10 8 9 9 9
4 3 3 3 3 4 2 2 — 1 — —
4 6 8 6 — — — — — — — —
*No reaction. †Mild reaction. ‡Moderate reaction. §Severe reaction.
●
grade 3 (severe reaction): severe fibroblast proliferation and collagen fiber homogenization
The criteria for scoring the vascular changes are as follows: ● ● ●
●
grade 0 (no reaction): no significant vascular proliferation grade 1 (mild reaction): the number of vascular structures in 1 high (40⫻) power field is ⬍25 grade 2 (moderate reaction): the number of vascular structures in 1 high (40⫻) power field is between 25 and 50 grade 3 (severe reaction): the number of vascular structures in 1 high (40⫻) power field is ⬎50
The presence of granulation tissue and foreign body giant cells were also described based on subjective single-blind evaluation and scored as present (1) or absent (0). Statistical analysis was performed using Friedman and Cochran tests for intragroup comparison (1, 4, and 8 weeks) and the Kruskal-Wallis test for intergroup comparison (1, 4, and 8 weeks). RESULTS Macroscopic examination showed that wound healing was satisfactory in all animals. The severity of inflammatory tissue reaction to the implanted materials is presented in Table I. Because of problems inherent in laboratory processing, one of the remaining specimens of guttapercha that was in the 8-week group had to be excluded. One-week observations for group 1 In this group, the intensity of inflammatory reaction was similar among the implant materials, and
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Fig. 1. Epiphany implant at the 1-week observation period. Edema, moderate vascular proliferation (blue arrows), and mild inflammatory infiltrate (red arrow) can be seen alongside the Teflon tube (hematoxylin-eosin, original magnification ⫻200).
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Fig. 3. Resilon implant at the 1-week observation period. Hyalinization (H) and fibroblast proliferation (F) can be seen around the material (hematoxylin-eosin, original magnification ⫻100).
Fig. 2. Gutta-percha implant at the 1-week observation period. Tissue next to material shows disintegration of tissue with inflammatory infiltration (red arrows) and vascular (blue arrows) and fibroblast proliferation (F; hematoxylin-eosin, original magnification ⫻200).
intragroup comparison revealed no statistical differences (P ⬎ .05). A peripheral reaction was in progress, with a moderate to severe inflammatory infiltration noted and exudation in the tissue. Edema, moderate vascular proliferation, and mild inflammatory infiltration were present along the Epiphany implant (Fig. 1). The areas in contact with guttapercha and Resilon showed a severe granulomatous reaction with inflammatory infiltration, vascular and fibroblast proliferation, and also hyalinization (Figs. 2 and 3). Granulation tissue was observed along the side of the empty Teflon tube.
Fig. 4. Epiphany implant at the 4-week observation period. Note the diminishing number of inflammatory cells and fibrosis (hematoxylin-eosin, original magnification ⫻100).
Four-week observations for group 2 The intensity of the tissue responses decreased in all implant materials and was scored as mild to moderate (Figs. 4 and 5). Intragroup comparison revealed no
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Fig. 5. Resilon implant at the 4-week observation period. The intensity of the reaction has diminished (hematoxylin-eosin, original magnification ⫻100).
Onay et al. e63
Fig. 7. Epiphany implant at the 8-week observation period. Slight inflammation (red arrows) with vascular proliferation can be seen (blue arrows; hematoxylin-eosin, original magnification ⫻200).
Fig. 8. Resilon implant at the 8-week observation period. The tissue located in immediate contact with the implant material is free of inflammation (hematoxylin-eosin, original magnification ⫻100).
Fig. 6. Gutta-percha implant at the 8-week observation period. The intensity of the reaction has diminished; however, there is fibrosis (F) and inflammation (red arrow) (hematoxylin-eosin, original magnification ⫻100).
statistical differences between the materials (P ⬎ .05). Some of these specimens exhibited a thin fibrous capsule, free of inflammatory cells. On the other hand, a severe granulomatous reaction with inflammatory infil-
OOOOE September 2007 0.05 0.05 0.05 0.05 ⬎ ⬎ ⬎ ⬎ P P P P .001 .05 .001 .05 ⬍ ⬍ ⬍ ⬍ P P P P .001 .05 .001 .05 ⬍ ⬍ ⬍ ⬍ P P P P 0.05 0.05 0.05 0.05 ⬎ ⬎ ⬎ ⬎
levels of significance were set at P ⬍ .05. *All
⬍ ⬍ ⬍ ⬍ Epiphany Resilon Gutta-percha Teflon
P P P P
.05 .01 .001 .05
P P P P ⬍ ⬍ ⬍ ⬍
.01 .001 .001 .001
P P P P
⬎ ⬎ ⬎ ⬎
0.05 0.05 0.05 0.05
P P P P
⬎ ⬎ ⬎ ⬎
0.05 0.05 0.05 0.05
P P P P
⬍ ⬍ ⬍ ⬍
.001 .001 .001 .001
P P P P
⬍ ⬍ ⬍ ⬍
.001 .001 .01 .01
P P P P
⬍ ⬍ ⬍ ⬍
.05 .001 .001 .05
P P P P
⬍ ⬍ ⬍ ⬍
.001 .001 .001 .001
P P P P
⬍ ⬍ ⬍ ⬍
.05 .05 .05 .05
P P P P
⬍ ⬍ ⬍ ⬍
.01 .01 .001 .05
P P P P
⬍ ⬍ ⬍ ⬍
.001 .001 .001 .001
P P P P
⬍ ⬍ ⬍ ⬍
.05 .01 .05 .05
P P P P
⬍ ⬍ ⬍ ⬍
.01 .01 .001 .05
P P P P
⬍ ⬍ ⬍ ⬍
.01 .01 .001 .05
P P P P
4-8 wk 1-8 wk 1-4 wk
Presence of foreign body giant cells
4-8 wk 1-8 wk
Fibroblast proliferation
1-4 wk 4-8 wk 1-8 wk
Vascular proliferation
1-4 wk 4-8 wk 1-8 wk
Mast-cell infiltration
1-4 wk 4-8 wk 1-8 wk 1-4 wk
Table II. Intergroup comparison of the periods of the study*
DISCUSSION This study has demonstrated that all the materials implanted into the dorsal connective tissue of rats promoted a moderate to severe inflammatory reaction at the 1-week period, which decreased with time. Connective tissue healing was remarkable for all experimental materials despite the moderate inflammatory reaction observed in only 1 Epiphany sample at the 8-week observation period. The initially moderate to severe inflammatory reaction took place for all experimental and control materials was probably the result of the surgical trauma.6,7 The subcutaneous implantation is considered as a suitable secondary test for evaluation of biocompatibility properties of restorative and endodontic materials. Multiple studies have evaluated sealer biocompatibility by using different implantation vehicles, such as polyethylene tubes,7,8 silicon tubes,6,9 dentin tubes,10 and Teflon tubes.11,12 In this study, Teflon tubes were used because of their inert nature and suitability for bringing a test material into contact with living tissue in a controlled and effective manner.13 Various factors determine the biocompatibility of a resin-based material, particularly the amount and nature of leachable components. Conventional composite resins contain a polymerizable organic matrix that consist of several comonomers (e.g., Bis-GMA, UDMA, EGDMA, TEGDMA, etc.) and various additives that function as coinitiators, stabilizers, or inhibitors.14 Costa et al.7 has shown a moderate to intense inflammatory reaction in many samples of 2 current adhesive resins when implanted into dorsal connective tissue of rats, even at 30 days. The irritative effect of these
1-8 wk
Presence of granulation tissue
Eight-week observations for group 3 The inflammatory reaction varied from none to slight in this period, and intragroup comparison revealed no statistical differences between the materials (P ⬎ .05). The total picture was of very slight inflammation and healing (Figs. 6, 7, and 8). It is noteworthy that neither foreign body giant cells nor granulation tissue were observed at this time period. All the implant materials appeared to be equally well tolerated. The intergroup comparison using the Kruskal-Wallis test with different parameters is summarized in Table II. The intergroup comparison reveals that statistically significant differences exist between the periods of the study, which presented a decrement in the intensity of the tissue responses with time.
1-4 wk
tration and vascular and fibroblast proliferation were seen in the tissues surrounding some of the gutta-percha and Resilon specimens. No foreign body giant cells were observed at this time period.
4-8 wk
Onay et al.
Stromal inflammatory cell infiltration
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materials was attributed to the unreacted monomers that remain after polymerization and induce a persistent local inflammatory reaction. Epiphany root canal sealer also contains a small percentage of BisGMA, ethoxylated BisGMA, urethane dimethacrylate, and hydrophilic difunctional methacrylates. The mild to severe inflammatory reaction at the 1-week period can possibly be attributed to these monomers, which could be displaced to connective tissue. Zmener6 reported on the implantation of a methacrylate-based endodontic sealer, so-called EndoRez (Ultradent Products, Inc., South Jordan, UT), into the connective tissues of rats. EndoRez and Epiphany sealers have similar formulation— urethane dimethacrylate and barium sulfate are common ingredients. The author reported connective tissue healing with time and also reported persistent inflammatory reactions in 2 of the EndoRez specimens, in contact with zinc and barium, from the 120-day observation period. This finding suggests the need for the development of further research with electron microprobe analysis to determine whether an occasional persistent breakdown of the sealer really disturb the periapical healing process. The intraosseous implant technique was used in guinea pigs by Sousa et al.,15 who reported that the Epiphany sealer was the only material that presented intraosseous biocompatibility among the EndoRez and AH Plus sealers. They also observed biocompatibility to Epiphany with bone formation and none to slight inflammatory reaction. These findings are in agreement with our study. Gutta-percha and Resilon can be assumed to be inert because these materials are stable core materials. In addition, the biocompatibility of these materials was to be expected. When the results for the Epiphany sealer were compared with gutta-percha and Resilon, the intensity of inflammatory reaction was similar, and intragroup comparison revealed no statistical differences for all the periods of the study. Merdad et al.16 assessed the cytotoxicity of the components of the Epiphany and Resilon obturation system, compared with the AH Plus sealer and guttapercha, by using the Millipore filter test. The authors reported that both set sealers (24 hours, 48 hours), gutta-percha and Resilon, were characterized by noncytotoxic responses. However, it has also been suggested that Resilon has better biocompatibility than .02 taper gutta-percha (Premier Dental Products Co., Plymouth Meeting, PA), .06 taper gutta-percha (DiaDent), and .06 taper Activ GP (Brasseler USA Dental Instrumentation, Savannah, GA) in an in vitro cell culture system by MTT assay (ATCC; American Type Culture Collection).17 According to the present
Onay et al. e65
study, it is shown that both the .04 taper gutta-percha and Resilon materials that also standardized in length had similar biocompatibility. Gutta-percha is the main filling material used in root canal treatment. The primary ingredient of a gutta-percha cone is zinc oxide (60%-70%), which provides a major part of the radiopacity. Gutta-percha accounts for approximately 20%, and the remaining ingredients are binders, opaquers, and coloring agents.18 Gutta-percha is considered to have acceptable biocompatibility with the formation of collagenous capsule around the implants, with very little or no inflammatory host response.19,20 In the present study, gutta-percha cones proved to have well-tolerated biocompatibility. The results of the present study demonstrate that all the implanted materials are well tolerated by tissues and have acceptable biocompatibility. However, before extrapolation of these results to the clinical situation in human beings, further studies are necessary to evaluate the suitability of the Epiphany sealer and Resilon points as an endodontic obturation system. REFERENCES 1. Nguyen TN. Obturation of the root canal system. In: Cohen S, Burns RC, editors. Pathways of the pulp. 3rd ed. St. Louis: CV Mosby; 1984. p. 205-15. 2. Schilder H. Filling the root canal in three dimensions. Dent Clin North Am 1967;11:723-44. 3. Orstavik D. Endodontic materials. Adv Dent Res 1988;2:12-24. 4. Teixeira FB, Teixeira ECN, Thompson JY, Trope M. Fracture resistance of roots endodontically treated with a new resin filling material. J Am Dent Assoc 2004;135:646-52. 5. Shipper G, Orstavik D, Teixeira FB, Trope M. An evaluation of microbial leakage in roots filled with a thermoplastic synthetic polymer-based root canal filling material (Resilon). J Endod 2004;30:342-7. 6. Zmener O. Tissue response to a new methacrylate-based root canal sealer: preliminary observations in the subcutaneous connective tissue of rats. J Endod 2004;30:348-51. 7. Costa CA, Teixeira HM, do Nascimento AB, Hebling J. Biocompatibility of two current adhesive resins. J Endod 2000;26:512-6. 8. Molloy D, Goldman M, White RR, Kabani S. Comparative tissue tolerance of a new endodontic sealer. Oral Surg Oral Med Oral Pathol 1992;73:490-3. 9. Kaplan AE, Ormaechea MF, Picca M, Canzobre MC, Ubios AM. Rheological properties and biocompatibility of endodontic sealers. Int Endod J 2003;36:527-32. 10. Holland R, de Souza V, Nery MJ, Bernabe PFE, Filho JAO, Dezan E Jr, et al. Calcium salt deposition in rat connective tissue after the implantation of calcium hydroxide-containing sealers. J Endod 2002;28:173-6. 11. Olsson B, Sliwkowski A, Langeland K. Subcutaneous implantation for the biological evaluation of endodontic material. J Endod 1981;7:355-69. 12. Kim JS, Baek SH, Bae KS. In vivo study on the biocompatibility of newly developed calcium phosphate-based root canal sealers J Endod 2004;30:708-11.
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13. Tronstad L, Barnett F, Flax M. Solubility and biocompatibility of calcium-hydroxide containing root canal sealers. Endod Dent Traumatol 1988;4:153-9. 14. Geurtsen W. Biocompatibility of resin-modified filling materials. Crit Rev Oral Biol Med 2000;11:333-55. 15. Sousa CJ, Montes CR, Pascon EA, Loyola AM, Versiani MA. Comparison of the intraosseous biocompatibility of AH Plus, EndoRez and Epiphany root canal sealers. J Endod 2006;32:656-62. 16. Merdad K, Pascon E, Kulkarni G, Santerre P, Friedman S. Cytotoxicity of Epiphany and Resilon™ assessed by the millipore filter test [abstract]. J Endod 2006;32:247. 17. Zhu Q, Jiang J, Safavi K, Spangberg L. Biocompatibility of Activ GP and Resilon™ cones on L929 cells in vitro [abstract]. J Endod 2006;32:248. 18. Spangberg L. Endodontic treatment of teeth without apical peri-
odontitis. In: Orstavik D, Pitt Ford TR, editors. Essential endodontology. 2nd ed. Cambridge, UK: Blackwell Science; 1999. p. 228. 19. Spangberg L. Biological effects of root-canal-filling materials. Part 7. Reaction of bony tissue to implanted root-canal-filling material in guinea pigs. Odontol Tidskr 1969;77:133-59. 20. Wolfson EM, Seltzer S. Reaction of rat connective tissue to some gutta-percha formulations. J Endod 1975;1:395-402. Reprint requests: Emel Olga Onay, DDS, PhD Faculty of Dentistry Baskent University 11. sokak No: 26 06490 Bahcelievler Ankara, Turkey [email protected]