MICROSCOPY RESEARCH AND TECHNIQUE 77:335–340 (2014)
In Vivo Biocompatibility Versus Degree of Conversion of Resin-Reinforced Cements in Different Time Periods ~ ROGERIO LACERDA-SANTOS,1* MARIA ISABEL SERPA SIMOES DE FARIAS,1 FABIOLA GALBIATTI DE CARVALHO,1 MATHEUS MELO PITHON,2 POLLIANNA MUNIZ ALVES,3 1 ^ ORLANDO MOTOHIRO TANAKA,4 AND GYMENNA MARIA TENORIO GUENES 1 Department of Orthodontics and Clinic, Federal University of Campina Grande, Paraıba, Brazil, Av. dos Universit arios, s/n, Rodovia Patos/Teixeira, Km1, Santa Cecılia, CEP 58700-970 2 Department of Orthodontics, State University of Sudoeste da Bahia, Bahia, Brazil, Rua Jose Moreira Sobrinho, s/n Jequiezinho, Jequie - BA, CEP 45200-000 3 Department of Patology, State University of Paraıba, Paraıba, Brazil, Rua Bara unas, 351, Bodocong o, Campina Grande - PB, CEP 58429-500 4 Department of Orthodontics, Pontifical Catholic University of Paran a, Paran a, Brazil, Rua Imaculada Conceic¸~ ao, 1155, Prado Velho, Curitiba - PR, CEP 81611-970
KEY WORDS
glass ionomer cements; biocompatibility; orthodontics
ABSTRACT This study focused on test the null hypothesis that there is no difference between the degree of conversion and biocompatibility of different resin reinforced glass ionomer cements (RRGICs). Forty-eight male Wistar rats were used, distributed into four groups (n 5 12), as follows: Group C (Control, polyethylene), Group FOB (Fuji Ortho Band), Group UBL (Ultra band Lok), and Group MCG (Multicure Glass), in subcutaneous tissue. The events of edema, necrosis, granulation tissue, multinuclear giant cells, young fibroblasts, and collagen formation were analyzed at 7, 15, and 30 days. The degree of conversion was evaluated by the Fourier method. Biocompatibility and degree of conversion were assessed using the Kruskal–Wallis and Dunn tests, and ANOVA and Tukey’s test, respectively (P < 0.05). It was observed that, there was significant difference between Groups FOB and UBL for the presence of young fibroblasts at 15 days (P 5 0.034) and between the Control and MCG Groups for the presence of multinucleated giant cells at 30 days (P 5 0.009). Monomer conversion increased progressively until day 30, with significant difference between Group FOB and Groups UBL and MCG (P 5 0.013) at 15 days. The null hypothesis was partially accepted, Fuji Ortho Band showed a less monomer conversion and a smaller number of young fibroblasts in the time of 15 days. Microsc. Res. Tech. 77:335–340, 2014. V 2014 Wiley Periodicals, Inc. C
INTRODUCTION Resin reinforced glass ionomer cements (RRGICs) have been used in a wide range of clinical applications (Mount, 1993), such as in orthodontic patients for bracket bonding and band cementation. On the other hand, substances commonly present in RRGICs, such as residual monomers, have been shown to have a defined cytotoxic (Costa et al., 1999; Kostoryz et al., 2003) and a similar behavior could occur in tissues, due to the proximity of orthodontic accessories to the gingiva and oral tissues (Gaintantzopoulou et al., 1994; Souza et al., 2006). RRGICs (de Souza Costa et al., 2003) have shown more cytotoxic effects than conventional ionomer cements, due to the quantity of leachable components from resins, such as 2-hydroxyethyl methacrylate (HEMA), frequently added to their chemical compositions (de Souza Costa et al., 2003; Geurtsen, 1998), with action on the dentinal tubules (Hashimoto et al., 2004), cell cultures (Hanks et al., 1991; Hanks et al., 1996), and gingival tissue (Santos et al., 2010). The tissue compatibility of these cements is directly linked to their chemical formulation and the release of monomers that are not converted into polymers after polymerization (Costa et al., 2000). Thus, residual C V
2014 WILEY PERIODICALS, INC.
monomers (Golin et al., 1992) may cause discrete to severe inflammatory reactions (Santos et al., 2010), and directly influences on the physical, mechanical and biological properties of the material (dos Santos et al., 2012; Shinya et al., 2009). The biocompatibility of materials (Costa et al., 2000; Santos et al., 2010) has been a concern. But there is a lack of studies that directly relate the degree of monomer conversion and the inflammatory and cure events of RRGICs in vivo. Taken as a set, the authors’ aim was to test the null hypothesis that there is no difference between the degree of conversion and biocompatibility of different RRGICs used for orthodontic band cementation at different time intervals.
*Correspondence to: Rogerio Lacerda-Santos, Federal University of Campina Grande - UFCG, Center for Health and Technology Rural - CSTR, Av. dos Universit arios, s/n, Rodovia Patos/Teixeira, Km1, Santa Cecılia, CEP: 58700-970, Patos, Paraıba, Brazil. E-mail: [email protected] or [email protected]. Received 28 September 2013; accepted in revised form 8 February 2014 REVIEW EDITOR: Dr. Chuanbin Mao Contract grant sponsor: National Council for Scientific and Technological Development-CNPq (PIBIC scholarship). DOI 10.1002/jemt.22348 Published online 25 February 2014 in Wiley Online Library (wileyonlinelibrary. com).
336 Groups FOB
UBL MCG
R. LACERDA-SANTOS ET AL. TABLE 1. Composition of the tested glass ionomer cements GICs/Composition “Fuji Ortho Band”: “Paste A”: 2-hydroxyethyl methacrylate (HEMA), Aluminum silicate glass. “Paste B”: Urethane dimethacrylate (UDMA) “Ultra Band Lok”: Components of glass, amorphous silica, HEMA, bisphenol A glycidylmethacrylate (Bis-GMA), sodium fluoride “Multi-Cure Glass Ionomer Band”: “Powder”: Fluoraluminosilicate glass. “Liquid”: Aqueous solution of polyalkenoic acid, HEMA
MATERIAL AND METHODS Animal Model and Experimental Groups Forty-eight adult male Wistar rats were used, with a mean weight of 250 g, divided into four experimental groups (12 rats per group): Group C (Control, polyethylene tube), Group FOB (Fuji Ortho Band), Group UBL (Ultra band Lok), and Group MCG (Multicure Glass) (Table 1). The rats were anesthetized with an intraperitoneal injection of sodium thiopental (50 mg/ kg) (Crist alia, S~ ao Paulo, Brazil). The experiment was approved by the Ethics Committee on Animal Research, CSTR\UFCG, CEP/No.092011. Trichotomy in the dorsal region and antisepsis of the operative field 4% chlorhexidine gluconate was used (Geurtsen, 1998). On the midline, equidistant from the insertion of the animal’s tale and head, two incisions approximately 8 mm long were made. The subcutaneous tissue was laterally parted to promote a tunnel (18 mm deep) in the lateral direction. Each rat received two tube implants (1.5 mm inner diameter 3 5 mm long) made of polyethylene (nontoxic Scalp Vein 19G) autoclaved at a temperature of 110 C for 20 min and then used as inoculation vehicles for the tested materials. Multicure glass ionomer was handled according to the manufacturers’ instructions, paper blocks, and a plastic spatula were previously autoclaved at a temperature of 110 C for 20 min. This procedure was not necessary for Fuji Ortho Band and Ultra Band Lok since the material was pre-measured and dispensed. After manipulation, the materials were introduced into the openings at the extremities of the tubes, using a syringe (Centrix, CT) supported on a glass slide at one extremity and a small glass slide at the other to flatten the material. After this, they were light polymerized for 40 s, using a LED appliance (Radii, SDI, Victoria, Australia) fixed on a rod to guarantee that the distance between the specimens, using a light intensity of 1000 mw/cm2, regularly calibrated with a radiometer (Model 100, Demetron Research, CT). After the polymerization of the RRGICs the tubes were implanted. The animals received 0.2 mL intramuscular dose of veterinary pentabiotic (Wyeth Laboratory, New York, NY), and an injection of sodium dipyrone (0.3 mL/100 g, Novalgina, S~ ao Paulo, Brazil). All the procedures of this study were performed in accordance with the Canadian Council on Animals Care (1981).
Lot/Manufacturer
304141 39460/GC American Corp., Tokyo, Japan
“116817/Reliance” Orthodontic Prod., Itasca, USA 8HA/8EU/3M Unitek, Monrovia, USA
After time intervals of 7, 15, and 30 days, the animals were sedated with sodium thiopental (50 mg/kg) (Crist alia) to obtain excisional biopsies of the implant area, including sufficient normal surrounding tissue. Each group consisted of 12 rats with two implants, resulting in 24 samples per group (eight samples of each material for each time intervals). The rats were sacrificed by the cervical dislocation technique. Biocompatibility The samples were taken and submitted to fixation in 4% formaldehyde for 24 h, and then embedded in paraffin to obtain serial histologic cuts 6 mm thick, and stained with hematoxylin and eosin. The inflammatory reaction induced by the RRGICs was evaluated by a blind examiner using a light microscope (BX40; Olympus, Hamburg, Germany) at 100, 200, and 4003 magnifications. The examiner was calibrated before data analysis (j 5 0.8). The cellular events with regard to the presence of inflammatory infiltrate, edema, necrosis, granulation tissue, multinuclear giant cells, young fibroblasts and collagen, were awarded points according to the following scores: 1, absent; 2, scarce; 3, moderate; and 4, intense. For each sample of the study, five representative sections of the histological condition of the tissue, when all five sections of the tissue showed the same histological condition, the scores 1, absent; 2, scarce; 3, moderate; and 4, intense represent: 1, absent (5.00); 2, scarce (10.00); 3, moderate (15.00); and 4, intense (20.00). Degree of Conversion Standardized test samples were prepared that measured 5 mm in diameter and 1.5 mm in thickness as follows: Stainless steel bipartite matrices were placed on a glass slide; the RRGICs were injected into them using a syringe (Centrix) and flattened with a small glass slide, followed by polymerization. A total of 45 test samples (n 5 5 per group) were stored in artificial saliva at 37 C in light proof boxes to prevent additional exposure to light. At 7, 15, and 30 days after curing and storage, each specimen was ground to obtain ionomer powder, which was subsequently mixed with potassium bromide (KBr) at a ratio (by weight) of 1:20. This powder was placed in a tablet maker under a pressure of approximately 8 tons. A spectrophotometer (Bomen-MB-102, Yukon, Canada) was used to evaluate the infrared spectrum measurements using the Fourier transformation method, to determine the degree of monomer conversion in percent. Microscopy Research and Technique
DEGREE OF CONVERSION OF RRGICS IN DIFFERENT TIME PERIODS
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TABLE 2. Mean and standard deviation of scores attributed to cements and control group, after time intervals of 7, 15, and 30 days Groups Event Inflammatory infiltrate Edema Necrosis Granulation tissue Giant cells Young fibroblasts Collagen
Time
FOB
UBL
MCG
C
P
7 days 15 days 30 days 7 days 15 days 30 days 7 days 15 days 30 days 7 days 15 days 30 days 7 days 15 days 30 days 7 days 15 days 30 days 7 days 15 days 30 days
15.00 (0.0) 15.00 (0.0) 12.50 (2.8) 15.00 (0.0) 7.50 (2.8) 5.00 (0.0) 6.25 (2.5) 5.00 (0.0) 5.00 (0.0) 16.25 (2.5) 13.75 (2.5) 10.00 (0.0) 5.00 (0.0) 6.25 (2.5) 7.50 (2.8)AB 8.75 (2.5) 12.50 (2.8)A 17.50 (2.8) 7.50 (2.8) 12.50 (2.8) 15.00 (0.0)
15.00 (0.0) 11.25 (2.5) 11.25 (2.5) 15.00 (0.0) 7.50 (2.8) 5.00 (0.0) 7.50 (2.8) 5.00 (0.0) 5.00 (0.0) 18.75 (2.5) 10.00 (0.0) 10.00 (0.0) 6.25 (2.5) 5.00 (0.0) 5.00 (0.0)A 10.00 (0.0) 20.00 (0.0)B 16.25 (2.5) 10.00 (0.0) 15.00 (0.0) 13.75 (2.5)
16.25 (2.5) 12.50 (2.8) 11.25 (2.5) 15.00 (0.0) 8.75 (2.5) 7.50 (2.8) 7.50 (2.8) 6.25 (2.5) 5.00 (0.0) 18.75 (2.5) 10.00 (0.0) 11.25 (2.5) 5.00 (0.0) 7.50 (2.8) 12.50 (2.8)B 10.00 (0.0) 17.50 (2.8)AB 16.25 (2.5) 10.00 (0.0) 15.00 (0.0) 13.75 (2.5)
13.75 (2.5) 10.00 (0.0) 10.00 (0.0) 16.25 (2.5) 5.00 (0.0) 5.00 (0.0) 6.25 (2.5) 5.00 (0.0) 5.00 (0.0) 18.75 (2.5) 7.50 (2.8) 12.50 (2.8) 6.25 (2.5) 6.25 (2.5) 5.00 (0.0)A 5.00 (0.0) 15.00 (0.0)AB 15.00 (0.0) 5.00 (0.0) 15.00 (0.0) 15.00 (0.0)
0.325 0.127 0.730 0.946 0.210 0.092 0.801 0.391 1.000 0.391 0.143 0.689 0.543 0.475 0.009 0.116 0.034 0.730 0.129 0.701 0.911
AB
Means followed by different letters express statistically significant difference (P < 0.05). Means followed by same letters not express significant differences (P > 0.05) according to non-parametric Kruskal–Wallis Test, followed by Dunn’s multiple comparisons test.
AA or BB
Statistical Analysis Statistical analysis was performed with BioEstat software program (version 5.0, Belem, Par a, Brazil). To evaluate biocompatibility, the cellular events were submitted to the Kruskall–Wallis nonparametric test, followed by Dunn’s test to determine the differences among the groups (P < 0.05), because they did not present normal distribution. The parametric data of the degree of materials conversion was submitted to the analysis of variance (ANOVA) followed by Tukey’s test (P < 0.05). RESULTS Biocompatibility From the 7th to 30th day, there was a gradual decrease in the inflammatory events, edema, and necrosis evaluated (Table 2). This reduction was less expressive in Group FOB for inflammatory infiltrate (mono- and polymorphonuclear cells) in comparison with the control group (Fig. 1A) and in Group MCG (Fig. 1B) for the circulatory alterations (edema) and tissue degeneration (necrosis) around and within the cavity, as a result of this cement implant. Although, there was no significant statistically difference between the cements (P > 0.05) for these three cellular events. On the other hand, the presence of granulation tissue and multinucleated giant cells that corresponding to a tissue response that indicates the onset of the repair process more intensively at 7 days, with 30 days was found to be more expressive in the Group MCG. Nevertheless, there was no significant difference between the cements evaluated in the 3 experimental time intervals (P > 0.05), except between the Group MCG, with UBL and Control groups for the presence of giant cells at the time interval of 30 days (P 5 0.009) (Table 2). There was a tendency for a more increased of young fibroblasts in Group UBL (Fig. 1C) and the presence of Microscopy Research and Technique
collagen found was similar for all groups, although the Groups UBL and MCG demonstrated largest amount of collagen (P 5 0.701) with 15 days. There was no significant difference for these events in the three experimental time intervals evaluated (P > 0.05), except between Group FOB (Fig. 1D) and Group UBL for the presence of young fibroblasts at the time interval of 15 days (P 5 0.034) (Table 2). After 30 days, in general, the control and experimental groups showed a chronic inflammatory process characterized by discrete events of mononuclear infiltrate, vascular alterations, formation of granulomas, young fibroblasts, and collagen formation around the samples. Degree of Conversion The monomer conversion of RRGICs increased progressively up to day 30, and was shown to be capable of complete conversion after this period. There were no significant difference between the cements at 7 and 30 days (P > 0.05). Fuji Ortho Band (Fig. 1D) cement showed the lowest conversion values at 15 and 30 days, with significant difference from the Ultra Band Lok (Fig. 1E) and Multicure Glass (Fig. 1F) cements (P 5 0.013) at the time interval of 15 days (Table 3). Histologically, it can be observed that with 15 days, Ultra Band Lok no showed multinucleated giant cells unlike other cements, and can evidence a greater amount of young fibroblasts in the Ultra Band Lok (Fig. 1E), when compared mainly with the Fuji Ortho Band cement. DISCUSSION Because the RRGICs used for cementation orthodontic bands come into intimate contact with the subgingival region, their compatibility with gingival tissues must be taken into account when they are being selected. The most studied method for in vivo analysis
Fig. 1. Photomicrograph of histological sample. A: 15 days after implantation, Control Group: shows evidence of edema (E) with scarce chronic inflammatory infiltrate (II) (HE, 2003 magnification; scale: 50 mm). B: Seven days after implantation, Group MCG: intense area of granulation reaction, with congested vessels (CV) and intense mononuclear inflammatory infiltrate (MI) (HE, 1003 magnification; scale: 100 mm). C: 30 days after implantation, Group UBL: intense fibroblast proliferation (FP), as well as moderate collagen fiber deposition (CFD), the majority of these being disposed in parallel bundles, indicating a repair process (HE, 2003 magnification; scale: 50 mm). D: 15 days after implantation, Group FOB: Moderate reaction of granulation tissue (GT), young fibroblasts (YF) and multinucleated
giant cells (GC), with nuclei arranged on the circular periphery (HE, 2003 magnification; scale: 50 mm). E: 15 days after implantation, Group UBL: Cavity of polyethylene tube surrounded by area of proliferation of fibroblasts (PF), blood vessels of small caliber (BV) and some areas of collagen fiber deposition (CFD) (HE, 4003 magnification; scale: 25 mm). F: 15 days after implantation, Group MCG: Low reaction of granulation tissue with blood vessels of small caliber (BV), and presence of multinucleated giant cells with nuclei distributed in the cell periphery (HE, 4003 magnification; scale: 25 mm). Area of polyethylene tube implant (PT). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
DEGREE OF CONVERSION OF RRGICS IN DIFFERENT TIME PERIODS TABLE 3. Mean values and standard deviation (SD) of the degree of conversion (%) of RRGICs Groups Times 7 days 15 days 30 days
FOB
UBL
MCG
P
85.3 (2.1) 89.5 (1.3)A 92.1 (1.7)
86.7 (2.9) 92.7 (2.3)B 94.0 (1.9)
83.1 (1.7) 92.2 (1.6)AB 94.1 (1.7)
0.102 0.013 0.379
P5 analysis of variance ANOVA and Tukey’s post hoc tests. AB Means followed by different letters express significant differences (P < 0.05). AA or BB Means followed by same letters not express significant differences (P > 0.05).
considers the inflammatory response (Santos et al., 2010). On the other hand, methacrylate monomers such as Bis-GMA (bisphenol A glycidylmethacrylate), UDMA (urethane dimethacrylate), and HEMA (2- hydroxyethyl methacrylate), used in the composition of RRGICs may cause cell damage (Ahrari et al., 2010; Costa et al., 1999; Hanks et al., 1991, 1996; Hashimoto et al., 2004; Kostoryz et al., 2003; Malkoc et al., 2010). Bis-GMA and UDMA are hydrophobic monomers frequently associated with HEMA. The diffusion of these monomers into tissues may be facilitated by HEMA, which increases the hydrophilic nature of the material. Under such conditions, the hydrophobic monomers may affect cells and cause tissue damage (de Souza et al., 2003; Gaintantzopoulou et al., 1994; Geurtsen, 1998; Hanks et al., 1991, 1996; Hashimoto et al., 2004; Souza et al., 2006). Since the intensity of the inflammatory process is related to the cytotoxicity of material and the decrease of inflammatory intensity relies on the control of individual defense system, which organizes itself to limit the aggressive action from the monomers, from local circulatory changes as edema (Costa et al., 2000; Golin et al., 1992) and favor the rapid tissue repair (Santos et al., 2010). Biocompatibility studies (Kaplan et al., 2003; Onay et al., 2007) have used polyethelyne tubes as controls (Grecca et al., 2011), as they are considered harmless to the epithelial and conjunctive tissue, in order to evaluate the inflammatory (Santos et al., 2010), and cure events caused by materials and, therefore, they were used in the present study. The initial inflammatory reaction events observed in this investigation at 7 days were more expressive for the RRGICs when compared with the Control. Inflammatory infiltrate, edema and necrosis demonstrated no significant difference between the materials and control group, indicating that the cements were well tolerated by the body. The Multi Cure cement demonstrated the most expressive inflammatory event at 7 days, supported by the lower monomer conversion shown in this period, in addition to which postpolymerization free polyalkenoic acids (Coimbra et al., 2006; Costa et al., 2003), may influence the inflammatory potential of these cements. Subsequently, small necrotic areas considered as the death of the tissue caused by factors that lead to irreversible cell damage (Santos et al., 2010; Golin et al., 1992), in association with edema surrounded by cell proliferation and young fibroblasts, subsequent deposition of collagen fibers and a reduction in the number of blood vessels were observed from the 15th to 30th Microscopy Research and Technique
339
days. The presence multinucleated giant cells at this stage is linked to the formation of granulomas, due to the polyethylene and/or cement, which was found to be more persistent in the group Multicure Glass with 30 days, although this did not significantly influenced the process of collagenization. The gradual decrease in inflammatory events and ascendency of the cure process at 30 days corroborates the findings of other studies (Grecca et al., 2011; Santos et al., 2010) and is related to the pattern of monomer conversion into polymers and consequent (Andrzejewska et al., 2003; dos Santos et al., 2012) release of residual monomers (Young et al., 2004) in the first 4 weeks (Santos et al., 2010). As described by Wan and Yap (1999), for the evaluation of monomer conversion (Eliades and Palaghias, 1993; Li et al., 1995; Palmer et al., 1999), the cement discs were ground and retransformed into discs with KBr, for evaluation of ionomeric cements in infrared spectrometry. RRGICs has two setting mechanisms: the acid–base reaction and/or the polymerization reaction of the monomer constituents (Palmer et al., 1999). The presence of potassium persulfate, an initiator of the acid-based polymerization reaction based on the redox system, which diminishes the quantity of residual monomers (Souza et al., 2006), and may explain the expressive monomer conversion of Multicure Glass at 30 days. The Fuji Ortho Band cement demonstrated the lowest values of conversion, added to this, the histologic results also showed greater expression in the inflammatory events and lower amount of young fibroblasts and collagen with 7 and 15 days, which shows the tendency of Fuji Ortho Band the a lower initial tissue repair response. On the other hand, Ultra band Lok cement demonstrated the highest value of conversion at the time interval of 15 days, histologically no showed multinucleated giant cells unlike other cements, and can evidence a greater amount of young fibroblasts in the Ultra Band Lok, when compared mainly with the Fuji Ortho Band cement. The results of this study are in agreement with those of other authors (Andrzejewska et al., 2003; Young et al., 2004) which have shown from 3 to 10% of residual monomers only, present after monomer conversion of RRGICs after 15 days. Nevertheless, the effects of the toxicity of resin components into tissues may be perceived for up to 2 years after polymerization (Tell et al., 1988). Analysis of the inflammatory and cure phenomena to characterize and classify experimental groups, comparing them with the control, allows one to affirm that Ultra Band Look, Fuji Ortho Band, and Multicure demonstrated a similar biologic behavior after 30 days, with the formation of chronic inflammation, dispersed multinucleated giant and collagen fibers. However, one cannot interpret the specificity of the data generated in animal models in vivo as a specific human response.
CONCLUSIONS The null hypothesis was partially accepted, Fuji Ortho Band showed a less monomer conversion and a smaller number of young fibroblasts in the time of 15 days. However, the cements showed to have similar
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biocompatibility in 30 days from the monomer conversion and reduction of aggression againt the tissue. REFERENCES Ahrari F, Tavakkol Afshari J, Poosti M, Brook A. 2010. Cytotoxicity of orthodontic bonding adhesive resins on human oral fibroblasts. Eur J Orthod 32:688–692. Andrzejewska E, Andrzejewski M, Socha E, Zych-Tomkowiak D. 2003. Effect of polyacid aqueous solutions on photocuring of polymerizable components of resin-modified glass ionomer cements. Dent Mater 19:501–509. Coimbra LR, Giro EMA, Aranha AMF, Costa CAS. 2006. Cytotoxicity of restorers glass ionomer cements on odontoblast lineage cell. Rev Odonto Ci^enc 21:338–345. Costa CA, Hebling J, Hanks CT. 2000. Current status of pulp capping with dentin adhesive systems: A review. Dent Mater 16:188–197. Costa CA, Teixeira HM, do Nascimento AB, Hebling J. 1999. Biocompatibility of an adhesive system and 2-hydroxyethylmethacrylate. ASDC J Dent Child 66:337–342. Costa CAS, Hebling J, Hanks CT. 2003. Effects of light-curing time on the cytotoxicity of a restorative resin composite applied to an immortalized odontoblast-cell line. Oper Dent 28:365–370. de Souza Costa CA, Hebling J, Garcia-Godoy F, Hanks CT. 2003. In vitro cytotoxicity of five glass-ionomer cements. Biomaterials 24: 3853–3858. dos Santos RL, Pithon MM, Martins FO, Romanos MT, Ruellas AC. 2012. Evaluation of cytotoxicity and degree of conversion of glass ionomer cements reinforced with resin. Eur J Orthod 34:362–366. Eliades G, Palaghias G. 1993. In vitro characterization of visible light-cured glass ionomer liners. Dent Mater 9:198–203. Gaintantzopoulou MD, Willis GP, Kafrawy AH. 1994. Pulp reactions to light-cured glass ionomer cements. Am J Dent 7:39–42. Geurtsen W. 1998. Substances released from dental resin composites and glass ionomer cements. Eur J Oral Sci 106:687–695. Golin C, Tavares T, Cunha AC. 1992. Biocompatibility evaluation of the trademarks of glass ionomer cement: Study in rat subcutaneous tissue. RBO 49:35–39. Grecca FS, Kopper PM, Santos RB, Fossati AC, Carrard VC, Acasigua GA, Figueiredo JA. 2011. Biocompatibility of RealSeal, its primer and AH Plus implanted in subcutaneous connective tissue of rats. J Appl Oral Sci 19:52–56. Hanks CT, Strawn SE, Wataha JC, Craig RG. 1991. Cytotoxic effects of resin components on cultured mammalian fibroblasts. J Dent Res 70:1450–1455.
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Microscopy Research and Technique
In Vivo Biocompatibility Versus Degree of Conversion of Resin-Reinforced Cements in Different Time Periods ~ ROGERIO LACERDA-SANTOS,1* MARIA ISABEL SERPA SIMOES DE FARIAS,1 FABIOLA GALBIATTI DE CARVALHO,1 MATHEUS MELO PITHON,2 POLLIANNA MUNIZ ALVES,3 1 ^ ORLANDO MOTOHIRO TANAKA,4 AND GYMENNA MARIA TENORIO GUENES 1 Department of Orthodontics and Clinic, Federal University of Campina Grande, Paraıba, Brazil, Av. dos Universit arios, s/n, Rodovia Patos/Teixeira, Km1, Santa Cecılia, CEP 58700-970 2 Department of Orthodontics, State University of Sudoeste da Bahia, Bahia, Brazil, Rua Jose Moreira Sobrinho, s/n Jequiezinho, Jequie - BA, CEP 45200-000 3 Department of Patology, State University of Paraıba, Paraıba, Brazil, Rua Bara unas, 351, Bodocong o, Campina Grande - PB, CEP 58429-500 4 Department of Orthodontics, Pontifical Catholic University of Paran a, Paran a, Brazil, Rua Imaculada Conceic¸~ ao, 1155, Prado Velho, Curitiba - PR, CEP 81611-970
KEY WORDS
glass ionomer cements; biocompatibility; orthodontics
ABSTRACT This study focused on test the null hypothesis that there is no difference between the degree of conversion and biocompatibility of different resin reinforced glass ionomer cements (RRGICs). Forty-eight male Wistar rats were used, distributed into four groups (n 5 12), as follows: Group C (Control, polyethylene), Group FOB (Fuji Ortho Band), Group UBL (Ultra band Lok), and Group MCG (Multicure Glass), in subcutaneous tissue. The events of edema, necrosis, granulation tissue, multinuclear giant cells, young fibroblasts, and collagen formation were analyzed at 7, 15, and 30 days. The degree of conversion was evaluated by the Fourier method. Biocompatibility and degree of conversion were assessed using the Kruskal–Wallis and Dunn tests, and ANOVA and Tukey’s test, respectively (P < 0.05). It was observed that, there was significant difference between Groups FOB and UBL for the presence of young fibroblasts at 15 days (P 5 0.034) and between the Control and MCG Groups for the presence of multinucleated giant cells at 30 days (P 5 0.009). Monomer conversion increased progressively until day 30, with significant difference between Group FOB and Groups UBL and MCG (P 5 0.013) at 15 days. The null hypothesis was partially accepted, Fuji Ortho Band showed a less monomer conversion and a smaller number of young fibroblasts in the time of 15 days. Microsc. Res. Tech. 77:335–340, 2014. V 2014 Wiley Periodicals, Inc. C
INTRODUCTION Resin reinforced glass ionomer cements (RRGICs) have been used in a wide range of clinical applications (Mount, 1993), such as in orthodontic patients for bracket bonding and band cementation. On the other hand, substances commonly present in RRGICs, such as residual monomers, have been shown to have a defined cytotoxic (Costa et al., 1999; Kostoryz et al., 2003) and a similar behavior could occur in tissues, due to the proximity of orthodontic accessories to the gingiva and oral tissues (Gaintantzopoulou et al., 1994; Souza et al., 2006). RRGICs (de Souza Costa et al., 2003) have shown more cytotoxic effects than conventional ionomer cements, due to the quantity of leachable components from resins, such as 2-hydroxyethyl methacrylate (HEMA), frequently added to their chemical compositions (de Souza Costa et al., 2003; Geurtsen, 1998), with action on the dentinal tubules (Hashimoto et al., 2004), cell cultures (Hanks et al., 1991; Hanks et al., 1996), and gingival tissue (Santos et al., 2010). The tissue compatibility of these cements is directly linked to their chemical formulation and the release of monomers that are not converted into polymers after polymerization (Costa et al., 2000). Thus, residual C V
2014 WILEY PERIODICALS, INC.
monomers (Golin et al., 1992) may cause discrete to severe inflammatory reactions (Santos et al., 2010), and directly influences on the physical, mechanical and biological properties of the material (dos Santos et al., 2012; Shinya et al., 2009). The biocompatibility of materials (Costa et al., 2000; Santos et al., 2010) has been a concern. But there is a lack of studies that directly relate the degree of monomer conversion and the inflammatory and cure events of RRGICs in vivo. Taken as a set, the authors’ aim was to test the null hypothesis that there is no difference between the degree of conversion and biocompatibility of different RRGICs used for orthodontic band cementation at different time intervals.
*Correspondence to: Rogerio Lacerda-Santos, Federal University of Campina Grande - UFCG, Center for Health and Technology Rural - CSTR, Av. dos Universit arios, s/n, Rodovia Patos/Teixeira, Km1, Santa Cecılia, CEP: 58700-970, Patos, Paraıba, Brazil. E-mail: [email protected] or [email protected]. Received 28 September 2013; accepted in revised form 8 February 2014 REVIEW EDITOR: Dr. Chuanbin Mao Contract grant sponsor: National Council for Scientific and Technological Development-CNPq (PIBIC scholarship). DOI 10.1002/jemt.22348 Published online 25 February 2014 in Wiley Online Library (wileyonlinelibrary. com).
336 Groups FOB
UBL MCG
R. LACERDA-SANTOS ET AL. TABLE 1. Composition of the tested glass ionomer cements GICs/Composition “Fuji Ortho Band”: “Paste A”: 2-hydroxyethyl methacrylate (HEMA), Aluminum silicate glass. “Paste B”: Urethane dimethacrylate (UDMA) “Ultra Band Lok”: Components of glass, amorphous silica, HEMA, bisphenol A glycidylmethacrylate (Bis-GMA), sodium fluoride “Multi-Cure Glass Ionomer Band”: “Powder”: Fluoraluminosilicate glass. “Liquid”: Aqueous solution of polyalkenoic acid, HEMA
MATERIAL AND METHODS Animal Model and Experimental Groups Forty-eight adult male Wistar rats were used, with a mean weight of 250 g, divided into four experimental groups (12 rats per group): Group C (Control, polyethylene tube), Group FOB (Fuji Ortho Band), Group UBL (Ultra band Lok), and Group MCG (Multicure Glass) (Table 1). The rats were anesthetized with an intraperitoneal injection of sodium thiopental (50 mg/ kg) (Crist alia, S~ ao Paulo, Brazil). The experiment was approved by the Ethics Committee on Animal Research, CSTR\UFCG, CEP/No.092011. Trichotomy in the dorsal region and antisepsis of the operative field 4% chlorhexidine gluconate was used (Geurtsen, 1998). On the midline, equidistant from the insertion of the animal’s tale and head, two incisions approximately 8 mm long were made. The subcutaneous tissue was laterally parted to promote a tunnel (18 mm deep) in the lateral direction. Each rat received two tube implants (1.5 mm inner diameter 3 5 mm long) made of polyethylene (nontoxic Scalp Vein 19G) autoclaved at a temperature of 110 C for 20 min and then used as inoculation vehicles for the tested materials. Multicure glass ionomer was handled according to the manufacturers’ instructions, paper blocks, and a plastic spatula were previously autoclaved at a temperature of 110 C for 20 min. This procedure was not necessary for Fuji Ortho Band and Ultra Band Lok since the material was pre-measured and dispensed. After manipulation, the materials were introduced into the openings at the extremities of the tubes, using a syringe (Centrix, CT) supported on a glass slide at one extremity and a small glass slide at the other to flatten the material. After this, they were light polymerized for 40 s, using a LED appliance (Radii, SDI, Victoria, Australia) fixed on a rod to guarantee that the distance between the specimens, using a light intensity of 1000 mw/cm2, regularly calibrated with a radiometer (Model 100, Demetron Research, CT). After the polymerization of the RRGICs the tubes were implanted. The animals received 0.2 mL intramuscular dose of veterinary pentabiotic (Wyeth Laboratory, New York, NY), and an injection of sodium dipyrone (0.3 mL/100 g, Novalgina, S~ ao Paulo, Brazil). All the procedures of this study were performed in accordance with the Canadian Council on Animals Care (1981).
Lot/Manufacturer
304141 39460/GC American Corp., Tokyo, Japan
“116817/Reliance” Orthodontic Prod., Itasca, USA 8HA/8EU/3M Unitek, Monrovia, USA
After time intervals of 7, 15, and 30 days, the animals were sedated with sodium thiopental (50 mg/kg) (Crist alia) to obtain excisional biopsies of the implant area, including sufficient normal surrounding tissue. Each group consisted of 12 rats with two implants, resulting in 24 samples per group (eight samples of each material for each time intervals). The rats were sacrificed by the cervical dislocation technique. Biocompatibility The samples were taken and submitted to fixation in 4% formaldehyde for 24 h, and then embedded in paraffin to obtain serial histologic cuts 6 mm thick, and stained with hematoxylin and eosin. The inflammatory reaction induced by the RRGICs was evaluated by a blind examiner using a light microscope (BX40; Olympus, Hamburg, Germany) at 100, 200, and 4003 magnifications. The examiner was calibrated before data analysis (j 5 0.8). The cellular events with regard to the presence of inflammatory infiltrate, edema, necrosis, granulation tissue, multinuclear giant cells, young fibroblasts and collagen, were awarded points according to the following scores: 1, absent; 2, scarce; 3, moderate; and 4, intense. For each sample of the study, five representative sections of the histological condition of the tissue, when all five sections of the tissue showed the same histological condition, the scores 1, absent; 2, scarce; 3, moderate; and 4, intense represent: 1, absent (5.00); 2, scarce (10.00); 3, moderate (15.00); and 4, intense (20.00). Degree of Conversion Standardized test samples were prepared that measured 5 mm in diameter and 1.5 mm in thickness as follows: Stainless steel bipartite matrices were placed on a glass slide; the RRGICs were injected into them using a syringe (Centrix) and flattened with a small glass slide, followed by polymerization. A total of 45 test samples (n 5 5 per group) were stored in artificial saliva at 37 C in light proof boxes to prevent additional exposure to light. At 7, 15, and 30 days after curing and storage, each specimen was ground to obtain ionomer powder, which was subsequently mixed with potassium bromide (KBr) at a ratio (by weight) of 1:20. This powder was placed in a tablet maker under a pressure of approximately 8 tons. A spectrophotometer (Bomen-MB-102, Yukon, Canada) was used to evaluate the infrared spectrum measurements using the Fourier transformation method, to determine the degree of monomer conversion in percent. Microscopy Research and Technique
DEGREE OF CONVERSION OF RRGICS IN DIFFERENT TIME PERIODS
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TABLE 2. Mean and standard deviation of scores attributed to cements and control group, after time intervals of 7, 15, and 30 days Groups Event Inflammatory infiltrate Edema Necrosis Granulation tissue Giant cells Young fibroblasts Collagen
Time
FOB
UBL
MCG
C
P
7 days 15 days 30 days 7 days 15 days 30 days 7 days 15 days 30 days 7 days 15 days 30 days 7 days 15 days 30 days 7 days 15 days 30 days 7 days 15 days 30 days
15.00 (0.0) 15.00 (0.0) 12.50 (2.8) 15.00 (0.0) 7.50 (2.8) 5.00 (0.0) 6.25 (2.5) 5.00 (0.0) 5.00 (0.0) 16.25 (2.5) 13.75 (2.5) 10.00 (0.0) 5.00 (0.0) 6.25 (2.5) 7.50 (2.8)AB 8.75 (2.5) 12.50 (2.8)A 17.50 (2.8) 7.50 (2.8) 12.50 (2.8) 15.00 (0.0)
15.00 (0.0) 11.25 (2.5) 11.25 (2.5) 15.00 (0.0) 7.50 (2.8) 5.00 (0.0) 7.50 (2.8) 5.00 (0.0) 5.00 (0.0) 18.75 (2.5) 10.00 (0.0) 10.00 (0.0) 6.25 (2.5) 5.00 (0.0) 5.00 (0.0)A 10.00 (0.0) 20.00 (0.0)B 16.25 (2.5) 10.00 (0.0) 15.00 (0.0) 13.75 (2.5)
16.25 (2.5) 12.50 (2.8) 11.25 (2.5) 15.00 (0.0) 8.75 (2.5) 7.50 (2.8) 7.50 (2.8) 6.25 (2.5) 5.00 (0.0) 18.75 (2.5) 10.00 (0.0) 11.25 (2.5) 5.00 (0.0) 7.50 (2.8) 12.50 (2.8)B 10.00 (0.0) 17.50 (2.8)AB 16.25 (2.5) 10.00 (0.0) 15.00 (0.0) 13.75 (2.5)
13.75 (2.5) 10.00 (0.0) 10.00 (0.0) 16.25 (2.5) 5.00 (0.0) 5.00 (0.0) 6.25 (2.5) 5.00 (0.0) 5.00 (0.0) 18.75 (2.5) 7.50 (2.8) 12.50 (2.8) 6.25 (2.5) 6.25 (2.5) 5.00 (0.0)A 5.00 (0.0) 15.00 (0.0)AB 15.00 (0.0) 5.00 (0.0) 15.00 (0.0) 15.00 (0.0)
0.325 0.127 0.730 0.946 0.210 0.092 0.801 0.391 1.000 0.391 0.143 0.689 0.543 0.475 0.009 0.116 0.034 0.730 0.129 0.701 0.911
AB
Means followed by different letters express statistically significant difference (P < 0.05). Means followed by same letters not express significant differences (P > 0.05) according to non-parametric Kruskal–Wallis Test, followed by Dunn’s multiple comparisons test.
AA or BB
Statistical Analysis Statistical analysis was performed with BioEstat software program (version 5.0, Belem, Par a, Brazil). To evaluate biocompatibility, the cellular events were submitted to the Kruskall–Wallis nonparametric test, followed by Dunn’s test to determine the differences among the groups (P < 0.05), because they did not present normal distribution. The parametric data of the degree of materials conversion was submitted to the analysis of variance (ANOVA) followed by Tukey’s test (P < 0.05). RESULTS Biocompatibility From the 7th to 30th day, there was a gradual decrease in the inflammatory events, edema, and necrosis evaluated (Table 2). This reduction was less expressive in Group FOB for inflammatory infiltrate (mono- and polymorphonuclear cells) in comparison with the control group (Fig. 1A) and in Group MCG (Fig. 1B) for the circulatory alterations (edema) and tissue degeneration (necrosis) around and within the cavity, as a result of this cement implant. Although, there was no significant statistically difference between the cements (P > 0.05) for these three cellular events. On the other hand, the presence of granulation tissue and multinucleated giant cells that corresponding to a tissue response that indicates the onset of the repair process more intensively at 7 days, with 30 days was found to be more expressive in the Group MCG. Nevertheless, there was no significant difference between the cements evaluated in the 3 experimental time intervals (P > 0.05), except between the Group MCG, with UBL and Control groups for the presence of giant cells at the time interval of 30 days (P 5 0.009) (Table 2). There was a tendency for a more increased of young fibroblasts in Group UBL (Fig. 1C) and the presence of Microscopy Research and Technique
collagen found was similar for all groups, although the Groups UBL and MCG demonstrated largest amount of collagen (P 5 0.701) with 15 days. There was no significant difference for these events in the three experimental time intervals evaluated (P > 0.05), except between Group FOB (Fig. 1D) and Group UBL for the presence of young fibroblasts at the time interval of 15 days (P 5 0.034) (Table 2). After 30 days, in general, the control and experimental groups showed a chronic inflammatory process characterized by discrete events of mononuclear infiltrate, vascular alterations, formation of granulomas, young fibroblasts, and collagen formation around the samples. Degree of Conversion The monomer conversion of RRGICs increased progressively up to day 30, and was shown to be capable of complete conversion after this period. There were no significant difference between the cements at 7 and 30 days (P > 0.05). Fuji Ortho Band (Fig. 1D) cement showed the lowest conversion values at 15 and 30 days, with significant difference from the Ultra Band Lok (Fig. 1E) and Multicure Glass (Fig. 1F) cements (P 5 0.013) at the time interval of 15 days (Table 3). Histologically, it can be observed that with 15 days, Ultra Band Lok no showed multinucleated giant cells unlike other cements, and can evidence a greater amount of young fibroblasts in the Ultra Band Lok (Fig. 1E), when compared mainly with the Fuji Ortho Band cement. DISCUSSION Because the RRGICs used for cementation orthodontic bands come into intimate contact with the subgingival region, their compatibility with gingival tissues must be taken into account when they are being selected. The most studied method for in vivo analysis
Fig. 1. Photomicrograph of histological sample. A: 15 days after implantation, Control Group: shows evidence of edema (E) with scarce chronic inflammatory infiltrate (II) (HE, 2003 magnification; scale: 50 mm). B: Seven days after implantation, Group MCG: intense area of granulation reaction, with congested vessels (CV) and intense mononuclear inflammatory infiltrate (MI) (HE, 1003 magnification; scale: 100 mm). C: 30 days after implantation, Group UBL: intense fibroblast proliferation (FP), as well as moderate collagen fiber deposition (CFD), the majority of these being disposed in parallel bundles, indicating a repair process (HE, 2003 magnification; scale: 50 mm). D: 15 days after implantation, Group FOB: Moderate reaction of granulation tissue (GT), young fibroblasts (YF) and multinucleated
giant cells (GC), with nuclei arranged on the circular periphery (HE, 2003 magnification; scale: 50 mm). E: 15 days after implantation, Group UBL: Cavity of polyethylene tube surrounded by area of proliferation of fibroblasts (PF), blood vessels of small caliber (BV) and some areas of collagen fiber deposition (CFD) (HE, 4003 magnification; scale: 25 mm). F: 15 days after implantation, Group MCG: Low reaction of granulation tissue with blood vessels of small caliber (BV), and presence of multinucleated giant cells with nuclei distributed in the cell periphery (HE, 4003 magnification; scale: 25 mm). Area of polyethylene tube implant (PT). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
DEGREE OF CONVERSION OF RRGICS IN DIFFERENT TIME PERIODS TABLE 3. Mean values and standard deviation (SD) of the degree of conversion (%) of RRGICs Groups Times 7 days 15 days 30 days
FOB
UBL
MCG
P
85.3 (2.1) 89.5 (1.3)A 92.1 (1.7)
86.7 (2.9) 92.7 (2.3)B 94.0 (1.9)
83.1 (1.7) 92.2 (1.6)AB 94.1 (1.7)
0.102 0.013 0.379
P5 analysis of variance ANOVA and Tukey’s post hoc tests. AB Means followed by different letters express significant differences (P < 0.05). AA or BB Means followed by same letters not express significant differences (P > 0.05).
considers the inflammatory response (Santos et al., 2010). On the other hand, methacrylate monomers such as Bis-GMA (bisphenol A glycidylmethacrylate), UDMA (urethane dimethacrylate), and HEMA (2- hydroxyethyl methacrylate), used in the composition of RRGICs may cause cell damage (Ahrari et al., 2010; Costa et al., 1999; Hanks et al., 1991, 1996; Hashimoto et al., 2004; Kostoryz et al., 2003; Malkoc et al., 2010). Bis-GMA and UDMA are hydrophobic monomers frequently associated with HEMA. The diffusion of these monomers into tissues may be facilitated by HEMA, which increases the hydrophilic nature of the material. Under such conditions, the hydrophobic monomers may affect cells and cause tissue damage (de Souza et al., 2003; Gaintantzopoulou et al., 1994; Geurtsen, 1998; Hanks et al., 1991, 1996; Hashimoto et al., 2004; Souza et al., 2006). Since the intensity of the inflammatory process is related to the cytotoxicity of material and the decrease of inflammatory intensity relies on the control of individual defense system, which organizes itself to limit the aggressive action from the monomers, from local circulatory changes as edema (Costa et al., 2000; Golin et al., 1992) and favor the rapid tissue repair (Santos et al., 2010). Biocompatibility studies (Kaplan et al., 2003; Onay et al., 2007) have used polyethelyne tubes as controls (Grecca et al., 2011), as they are considered harmless to the epithelial and conjunctive tissue, in order to evaluate the inflammatory (Santos et al., 2010), and cure events caused by materials and, therefore, they were used in the present study. The initial inflammatory reaction events observed in this investigation at 7 days were more expressive for the RRGICs when compared with the Control. Inflammatory infiltrate, edema and necrosis demonstrated no significant difference between the materials and control group, indicating that the cements were well tolerated by the body. The Multi Cure cement demonstrated the most expressive inflammatory event at 7 days, supported by the lower monomer conversion shown in this period, in addition to which postpolymerization free polyalkenoic acids (Coimbra et al., 2006; Costa et al., 2003), may influence the inflammatory potential of these cements. Subsequently, small necrotic areas considered as the death of the tissue caused by factors that lead to irreversible cell damage (Santos et al., 2010; Golin et al., 1992), in association with edema surrounded by cell proliferation and young fibroblasts, subsequent deposition of collagen fibers and a reduction in the number of blood vessels were observed from the 15th to 30th Microscopy Research and Technique
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days. The presence multinucleated giant cells at this stage is linked to the formation of granulomas, due to the polyethylene and/or cement, which was found to be more persistent in the group Multicure Glass with 30 days, although this did not significantly influenced the process of collagenization. The gradual decrease in inflammatory events and ascendency of the cure process at 30 days corroborates the findings of other studies (Grecca et al., 2011; Santos et al., 2010) and is related to the pattern of monomer conversion into polymers and consequent (Andrzejewska et al., 2003; dos Santos et al., 2012) release of residual monomers (Young et al., 2004) in the first 4 weeks (Santos et al., 2010). As described by Wan and Yap (1999), for the evaluation of monomer conversion (Eliades and Palaghias, 1993; Li et al., 1995; Palmer et al., 1999), the cement discs were ground and retransformed into discs with KBr, for evaluation of ionomeric cements in infrared spectrometry. RRGICs has two setting mechanisms: the acid–base reaction and/or the polymerization reaction of the monomer constituents (Palmer et al., 1999). The presence of potassium persulfate, an initiator of the acid-based polymerization reaction based on the redox system, which diminishes the quantity of residual monomers (Souza et al., 2006), and may explain the expressive monomer conversion of Multicure Glass at 30 days. The Fuji Ortho Band cement demonstrated the lowest values of conversion, added to this, the histologic results also showed greater expression in the inflammatory events and lower amount of young fibroblasts and collagen with 7 and 15 days, which shows the tendency of Fuji Ortho Band the a lower initial tissue repair response. On the other hand, Ultra band Lok cement demonstrated the highest value of conversion at the time interval of 15 days, histologically no showed multinucleated giant cells unlike other cements, and can evidence a greater amount of young fibroblasts in the Ultra Band Lok, when compared mainly with the Fuji Ortho Band cement. The results of this study are in agreement with those of other authors (Andrzejewska et al., 2003; Young et al., 2004) which have shown from 3 to 10% of residual monomers only, present after monomer conversion of RRGICs after 15 days. Nevertheless, the effects of the toxicity of resin components into tissues may be perceived for up to 2 years after polymerization (Tell et al., 1988). Analysis of the inflammatory and cure phenomena to characterize and classify experimental groups, comparing them with the control, allows one to affirm that Ultra Band Look, Fuji Ortho Band, and Multicure demonstrated a similar biologic behavior after 30 days, with the formation of chronic inflammation, dispersed multinucleated giant and collagen fibers. However, one cannot interpret the specificity of the data generated in animal models in vivo as a specific human response.
CONCLUSIONS The null hypothesis was partially accepted, Fuji Ortho Band showed a less monomer conversion and a smaller number of young fibroblasts in the time of 15 days. However, the cements showed to have similar
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