The Effect of the Bleaching Agent Sodium Perborate on Macrophage Adhesion in Vitro: Implications in External Cervical Root Resorption

Alicia Jim(~nez-Rubio, DDS, MD, PhD, and Juan J. Segura, DDS, MD, PhD

The purpose of this study was to investigate the in The development of external cervical root resorption after in- vitro effect of sodium perborate, which is used as ternal bleaching of discolored pulpless teeth is a serious sequela a bleaching agent in the treatment of discolored that has been reported in the literature. External cervical root pulpless teeth, on substrate adherence capacity of resorption has been associated with the use of hydrogen peroxide, macrophages. Inflammatory macrophages were and a role for sodium perborate could not be excluded (7). One explanation given for this phenomenon is that it results from an obtained from Wistar rats and resuspended in inflammatory process initiated by the presence of bleaching agents RPMI-1640 medium. As a test of macrophage ad- in the attachment apparatus (8). hesion, the adherence capacity of macrophages to On the other hand, it has been demonstrated that inflamed a plastic surface was determined. Assays were periodontal tissues contain a variety of immunocompetent cells, conducted in Eppendorf tubes for 15 min of incu- with macrophages predominating (9). Several studies suggest that bation at 37°C in a humidified atmosphere of 5% macrophages and osteoclasts (macrophage-derived cells) play a C02. The adherence index was calculated. Results main role in the pathogenesis of external cervical root resorption. showed that sodium perborate decreased in a Macrophages stimulate osteoclastic bone resorption, and dentin dose-dependent manner and decreased signifi- and cementum destruction by the activation of litic processes in cantly (p < 0.05) the adherence index of rat perito- periodontal tissues (10-12). Moreover, macrophage-derived in- neal macrophages. Sodium perborate was less po- flammatory mediator--such as prostaglandin E 2, tumor necrosis factor-alpha, and interleukin-1/3----were found to be associated with tent than sodium hypochlorite and eugenol in periodontal destruction and cervical root resorption (13). inhibiting macrophage adhesion. The inhibitory ef- Although molecular mechanisms involved in macrophage func- fect of sodium perborate on macrophage adhesion tion are poorly defined (14), it is well known that adherence is the further supports the concept that this agent is not first step in the phagocytic process of inflammatory macrophages implicated in external cervical root resorption as- (15). sociated with intracoronal bleaching. The effect of sodium perborate on macrophage phagocytic function has not been studied. In this study, the in vitro effect of sodium perborate on substrate adherence capacity of rat inflammatory macrophages was examined. Discoloration of nonvital teeth is an aesthetic deficiency frequently requiring treatment. The most common cause of tooth discolora- MATERIALS AND METHODS tion is intracoronal blood decomposition (1). Intracoronal bleaching is commonly used for the treatment of discolored endodonti- Chemic~s cally treated teeth due to its efficacy, simplicity, and relatively low cost (2). Several bleaching materials have been used for treating Sodium perborate was obtained from Oral-B Laboratorios discolored nonvital teeth. Traditionally, an aqueous solution of 30 (Amosan) (C/Caleruega 102, Madrid, Spain), and RPMI-1640 me- to 35% hydrogen peroxide and heat were used for this purpose. dium was obtained from Sigma Chemical Co. (St. Louis, MO). All Later, hydrogen peroxide was used instead of water to obtain other chemicals were reagent grade. synergism and enhance the bleaching efficacy (3). Salvas (4) reported successful results using sodium perborate as a bleaching agent. Spasser (5) bleached teeth successfully without hydrogen Collection of Rat Peritoneal Macrophages peroxide or heat using a paste of sodium perborate and water. Sodium perborate is frequently used as a bleaching agent in the The protocol was approved by our experimentation committee. walkingbleach technique and mixed with hydrogen peroxide in the Peritoneal macrophages were elicited from Wistar rats by the thermocatalytic method (6). method described previously (16). Briefly, each rat was injected 229 230 Jim(~nez-Rubio and Segura Journal of Endodontics intraperitoneally with 5 ml of sterile 6% sodium caseinate. Ani- mals were killed after 4 days by decapitation, and the peritoneal 70 cavity was washed with 10 ml of cold 0.9% NaC1. After a 2-min massage, the cell exudate was removed with a syringe and centri- x 60 fuged for 10 min at 250 × g at 4°C. The contaminating red blood cells were lysed with cold 0.2% NaC1. The remaining cells were 5o then washed with 0.9% NaC1 by centrifugation, resuspended in RPMI-1640 medium, counted, adjusted in the same medium at 8 40 c- 2-4 x 10 6 macrophages/ml and used immediately for experiments. Mean cells per rat varied from 20-30 × 10 6, of which 85 30 to 95% were macrophages by morphological criteria in Giemsa and Papanicolaou staining techniques. Viability, as determined by 20 trypan blue exclusion, was always >95%. 10 Assay of Substrate Adherence Capacity 0 0 15 30 The quantification of substrate adherence capacity was con- Time (min) ducted according to the technique described previously by de la Fuente et al. (17), with minor modifications. Aliquots of 180 p.1 of FIG 1. Time course of sodium perborate-inhibition of the AI of inflammatory macrophages. Macrophages (2-4 × 106/ml) were incu- cell suspension were dispensed in Eppendorf tubes that resemble bated at 37°C in a humidified atmosphere of 5% CO2 in the absence the adherence to tissues as reported by Noga et al. (18). Amosan ((3) or presence of increasing concentrations of sodium perborate (20/xl) was added to a final dilution of 1:10, 1:100, or 1:1000 in (0.0001 dilution, O; 0.001 dilution • and 0.01 dilution A). After 5, 15, the incubation medium. RPMI-1640 medium (20/xl) was added to or 30 min, the reaction was stopped and the AI calculated. Each control samples. Adherence assays were performed at 15 min of point is the mean of three separated experiments performed in incubation at 37°C in a humidified atmosphere of 5% CO 2 to duplicate. provide a maximal adherence index (19). After gentle removal (5 s in the vortex in position 5) of non-adherent cells, aliquots of 10 /zl from each sample were taken and the number of non-adherent 0.05 and p < 0.01, respectively). In subsequent experiments, macrophages/milliliter was counted in Neubauer chambers. No incubation of cells was performed at 37°C for 15 min in a humid- agglutination of macrophages was observed. When two substances ified atmosphere of 5% CO 2 to provide a maximal inhibitory effect were tested simultaneously, 10/xl of each were added together with of sodium perborate. 180/xl of cell suspension to the Eppendorf tubes, to a final volume The effect of sodium perborate on the AI of macrophages was of 200 ~1. The adherence index (AI) was calculated according to compared with the effect of sodium hypochlorite, another bleach- the following equation: ing agent, and eugenol (Fig. 2). As can be seen, when sodium perborate was added to the incubation medium at a final dilution of Nonadherent macrophages/ml 1:1000, a light decrease of AI was found (4.57%) (p > 0.05). AI = 100- X 100. Initial macrophages/ml However, lower sodium perborate dilutions (1:100 and 1:I0) decreased strongly and significantly the AI by 16% (p < 0.05) and 42.7%, respectively (p < 0.05). A 1:100 dilution of eugenol and Statistical Analysis sodium hypochlorite decreased significantly (p < 0.05) the AI of macrophages with more potency than sodium perborate. Eugenol All values were expressed as the mean - SEM of the number and sodium hypochlorite, at 1:10 dilution, virtually abolish the AI of experiments, performed in duplicate, as indicated in the corre- of macrophages. sponding figures. Data were evaluated statistically by Student's t test. A value of p < 0.05 (two-tailed) was considered statistically DISCUSSION significant.

In the present study, we demonstrated that sodium perborate RESULTS inhibits in vitro substrate adherence capacity of rat peritoneal macrophages. As far as we know, this is the first time that an effect Sodium perborate inhibited substrate adherence capacity of of sodium perborate on macrophages has been described. macrophages in all conditions tested. The inhibitory effect of The sensitivity of cells to a sodium perborate dilution as high as sodium perborate was a time- and dose-dependent phenomenon. 1:100, a concentration of sodium perborate lower than that used in The AIs obtained in control peritoneal macrophages and incubated the treatment of periodontal disease, and the potent inhibitory with different dilutions of sodium perborate (1:1000, 1:100, and effect of the lower dilution (1:10)--which is a sodium perborate 1:10) for 5, 15, and 30 min are shown in Fig. 1. As can be seen, concentration similar to that which could be found in gingival the substrate adherence capacity of control macrophages increased tissues--suggest that its inhibitory effect on macrophage substrate progressively, reaching a maximum between 15 and 30 min. When adherence capacity may have physiological significance in vivo at 1:1000 dilution of sodium perborate was added to the incubation the level of periodontal tissues. medium, no significative changes were found (p < 0.05). How- The phagocytic cell adherence to a smooth plastic surface is ever, higher sodium perborate concentrations (1:100 and 1:10 comparable with that taking place in animal tissues (17, 18). dilutions) decreased the AI significantly at all times tested (p < Taking into account that adhesion is the first step in the phagocytic Vol. 24, No. 4, April 1998 Perborate-Macrophage Adhesion 231

were associated with the use of Superoxol and heat during bleach- 100 ing (2). These reports suggest that bleaching-related resorption is [] PER provoked by highly concentrated hydrogen peroxide. On the other [] EUG hand, Holmstrup et al. (6) did not find any signs indicating cervical 80 I~HIP root resorption in teeth bleached with sodium perborate and water after a 3-yr follow-up. It is recognized that the bleaching efficacy of sodium perborate 6o is enhanced by the addition of 30% hydrogen peroxide (3). How- ever, an equal bleaching result may be accomplished without hydrogen peroxide by increasing the number of treatments (2). 1-® 40 Therefore, is recommended that sodium perborate be used mixed with water rather than with hydrogen peroxide to prevent the 20 occurrence of bleaching-associated external root resorption. We conclude that, taking into account that adhesion is the first step in the phagocytic function of macrophages and that osteoclasts o C 0.001 0.01 0.1 are macrophage-derived cells, the inhibitory effect of sodium perborate on macrophage adhesion could explain, at least in part, the Dilution infrequent cases of external cervical root resorption that occur FIG 2. Effect of sodium perborate (PER), sodium hypochlorite (HIP), when bleaching is performed only with sodium perborate. When- and eugenol (EUG) on the AI of rat peritoneal macrophages. Mac- ever indicated, bleaching with sodium perborate should be the rophages (2-4 × 106/ml) were incubated at 37°C in a humidified treatment of choice. atmosphere of 5% CO2 in the absence (control) or presence of increasing concentrations of sodium perborate, sodium hypochlo- This study was supported by grants from DGICYT (PB94-1434 and PM95- rite, and eugenol (0.0001,0.001, and 0.01 dilutions). After 15 min the 0159; Ministry of Education and Culture of Spain). reaction was stopped and the AI calculated. Each point is the mean Dr. Jim~nez-Rubio is affiliated with the Department of Dental Pathology of three separated experiments performed in duplicate. and Therapeutics, School of Dentistry; and Drs. Segura and Jim~nez-Rubio are both affiliated with the Department of Medical Biochemistry and Molecular Biology, School of Medicine and Dentistry, University of Seville, Seville, Spain. process and in antigen presentation (14, 15), sodium perborate Address requests for reprints to Dr. Juan J. Segura, c/Malign, 5, I°D, 41018 could inhibit phagocytosis in macrophages and reduce inflamma- Seville, Spain. tory reactions in gingival and periodontal tissues. Moreover, the sodium perborate concentration found in periodontal tissues after its use as bleaching agent corresponds to the concentration of References sodium perborate that caused maximal inhibition of macrophage AI. 1. 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