Although dental implants are currently

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1 Laser Therapy as an Effective Method for Implant Surface Decontamination: A Histomorphometric Study in Rats Samira Salmeron,* Maria L.R. Rezende,* Alberto Consolaro, Adriana C.P. Sant Ana,* Carla A. Damante,* Sebastião L.A. Greghi,* and Euloir Passanezi* Background: To the best of the authors knowledge, a standard protocol for treating peri-implantitis is not yet established. Methods: A total of 150 titanium disks with smooth or rough surfaces contaminated with microbial biofilm were implanted subcutaneously in rats after undergoing one of three treatments: 1) low-intensity laser (LIL); 2) antimicrobial photodynamic therapy (apdt); or 3) toluidine blue O (TBO). Sterile and contaminated disks served as negative (NC) and positive (C) control groups, respectively. After days 7, 28, and 84, tissue inflammation was evaluated microscopically by measuring the density of collagen fibers (degree of fibrosis) and concentration of polymorphonuclear neutrophils. Results: Surface texture did not affect the degree of inflammation, but the area of reactive tissue was significantly greater for rough implants ( µm 2 ) than for smooth ones ( µm 2 ; P = ). Group C presented the lowest and group NC presented the highest degree of fibrosis with significance only after day 7; these groups had the highest and lowest scores, respectively, for degree of inflammation. Group C showed the largest area of reactive tissue ( µm 2 ), but it was not significantly larger than group LIL (P = ) and group TBO (P = ). Group apdt showed the smallest area ( µm 2 ) of reactive tissue among the treatment groups. After day 28, groups LIL, apdt, TBO, and C resembled group NC in all the studied parameters. Conclusion: Group apdt showed more favorable results in parameter area of reactive tissue than the other methods after day 7, but over longer time periods all methods produced outcomes equivalent to sterile implants. J Periodontol 2013;84: KEY WORDS Anti-infective agents; decontamination; dental implants; lasers; peri-implantitis; wound healing. * Department of Prosthodontics, Division of Periodontics, Bauru School of Dentistry, University of São Paulo, Bauru, São Paulo, Brazil. Department of Stomatology, Division of Pathology, Bauru School of Dentistry, University of São Paulo. Although dental implants are currently a standard and common treatment, 1 with the gradual increase in the number of individuals rehabilitated with this approach, a gradual increase in peri-implant tissue complications are expected in the near future. 2 Microbiologic studies have shown that implant surfaces commonly harbor contaminants, especially lipopolysaccharides (LPSs) 3,4 that, when released, intensify the inflammatory responses while changing the superficial oxide layer. 5,6 These changes depend on various physical and biologic factors. For example, when exposed to the oral environment by tissue loss due to periimplantitis, rough-surfaced implants seem to have affected the removal of microorganisms by saliva flow and oral hygiene procedures. 7 There is evidence that microbial biofilms adhere more strongly to rough-surfaced implants than to smooth ones Several methods have been developed to decontaminate implant surfaces and to rescue peri-implant support tissues. These methods include chemical conditioning, topical or systemic antibiotic therapy, and laser application The goal of these methods is not only to remove the superficial contaminated oxide layer, keeping unmodified the implant surface topography, but also to ensure the integrity and regeneration of surrounding tissues Mechanical instrumentation doi: /jop

2 Low-intensity Laser, apdt, and TBO Decontaminate Implant Surfaces Volume 84 Number 5 with standard periodontal scalers can damage the surface. 20 Blasting with abrasive powders, although successful in vitro, has various drawbacks when applied in the clinical setting, such as microscopic alterations of the implant surface, increased risk of emphysema, 21 and the presence of residues on the surface, which is also the case with polytetrafluoroethylene scalers. 22 These limitations, as well as the challenge of accessing very deep and narrow bone defects, 23 have intensified the search for alternative treatments. Nevertheless, to the best of our knowledge, a standard protocol for treating periimplantitis has not yet been established. Several studies have reported viable methods for disinfecting implant surfaces with different kinds of low-intensity lasers (LIL), 6,16,22,24-27 antimicrobial photodynamic therapy (apdt), and dyes such as toluidine blue O (TBO) that have cytotoxic effects once attached to the bacterial cell wall. 32,35 These methods are commonly used because they have proven benefits in the treatment of peri-implantitis. 36 Moreover, they do not present drawbacks such as post-surgery complications, 13,17 changes in surface topography, 15,16 and the presence of remnants on the implant surfaces. 22 However, no studies were found comparing the effects of the previously mentioned methods on implants contaminated with a naturally deposited microbial biofilm and the tissue reaction following their implantation in living tissue. In this in vivo study, the intensity of the inflammatory infiltrate and degree of fibrosis produced in the subcutaneous connective tissue of rats by the implantation of contaminated titanium disks were compared after the disks were decontaminated with LIL, apdt, or TBO, to determine which one allows the biocompatibility recovery of the implant surface. MATERIALS AND METHODS This research was approved by the Ethical Committee on Human Research (permit no. 121/2008) and by the Ethical Committee on Animal Research (permit no. 015/2008) of the Bauru School of Dentistry, University of São Paulo, where the research was performed from July 2009 to March The study used the criteria for assessing tissue reactions to implants established by the ISO (1994). A total of 150 commercially pure titanium disks, measuring 1.5 mm thick and 4.0 mm in diameter and specially produced for the experiment, were used. Half of the disks (75) had smooth (polished) surfaces, and the other half had surfaces that were sandblasted, large grit, and acid-etched. Contamination and Decontamination of the Disks Thirty disks were kept sterile, served as negative controls, and were divided into 15 smooth-surfaced Figure 1. A) Removable acrylic palatal plates with previously prepared niches in which the titanium disks were placed underneath the nylon grid. B) Eight titanium disks (four smooth-surfaced disks on the right side and four rough-surfaced disks on the left side) protected by the nylon glued on the acrylic plate ready to be used by the volunteers. C) Aspect of the disks at the moment of removal from the niches evidencing bacterial biofilm formed on its surface. D) Study design. NC = negative control; C = positive control. disks and 15 rough-surfaced disks. A microbial biofilm was allowed to grow on the remaining 120 disks according to a methodology adapted from Furlani et al. 37 After signing an informed consent form, 15 non-smoker volunteers of both sexes, who were not using any medication and were without sites of gingival bleeding on probing, wore for 7 consecutive days a removable acrylic palatal plate in which niches were produced to lodge eight disks (four smooth-surfaced on the right side and four rough-surfaced on the left side). The disks were protected by a nylon grid glued i to the acrylic (Figs. 1A and 1B). Volunteers were instructed to remove the plates during meals and during oral hygiene, avoiding cleaning the grid or the disks. After day 7, the disks were removed from the plates(fig.1c).toremovethevisiblebiofilm,90of the disks were brushed with sterile saline solution, using five brushing movements on each surface of the disks. The remaining 30 disks were not brushed and served as positive controls divided into 15 smoothsurfaced (group C S ) and 15 rough-surfaced disks (group C R ). The brushed disks were transferred individually to wells of cell culture plates, # where they were divided into five groups consisting of 30 disks each (15 smooth and 15 rough) allocated to receive Titanium Fix, AS Technology, São José dos Campos, São Paulo, Brazil. Tela Fix, Ciplás, Rio de Janeiro, Rio de Janeiro, Brazil. i Super Bonder, Henkel, Itapevi, São Paulo, Brazil. Colgate-Palmolive, São Paulo, São Paulo, Brazil. # Biosystems, Curitiba, Paraná, Brazil. 642

3 Salmeron, Rezende, Consolaro, et al. one of the decontamination methods described herein (study design shown in Fig. 1D). LIL. An indium gallium aluminum phosphide** red laser-emitting apparatus was adjusted to 660-nm wavelength, 30 mw, and 45 J/cm 2 in continuous mode. With 600-mm-diameter fiber optics, the 30 disks of this group received perpendicular irradiation for 60 seconds distributed as horizontal (10 seconds), vertical (10 seconds), and oblique (10 seconds) sweeping on each side. Then, the disks were transferred to sterile glass ampoules kept sealed until their implantation in the subcutaneous tissue of rats. apdt. The disks were immersed in 1 ml TBO solution (at 100 mg/ml) poured with an automatic pipette into the wells. After 60 seconds, the same laser irradiation protocol was applied as for the LIL group. Immediately afterward, the diskswerestoredinsterile glass ampoules until their implantation in the rats. TBO. The disks were immersed for 60 seconds in 1 ml TBO solution in the same fashion as for the apdt group. Immediately after removal from TBO, the disks were stored in sterile glass ampoules until their implantation in the rats. Surgical Procedure A total of 150 adult male isogenic Wistar rats (Rattus norvegicus) weighing400gwereused.therats were kept in separate cages at a constant temperature and humidity (21 C and 72%, respectively) and were randomly selected for receiving one disk of each group. Surgical procedures were carried out under aseptic conditions. Rats were anesthetized with an intramuscular injection of 3 ml xylazine hydrochloride plus 10 ml ketamine hydrochloride. On the shaved dorsal region, an incision was made with a no. 15 blade ii to create an envelope where the titanium disk was inserted. The incision was closed with a 4.0 silk suture that was removed after day 7. Rats were offered water and food ## ad libitum. After days 7, 28, and 84, five animals of each group were sacrificed with an overdose of anesthesia, and tissue blocks containing the disks were removed and fixed in a 10% aqueous formaldehyde solution*** for 12 hours. Histology and Microscopic Analysis After fixation, specimens were cut in the middle to remove the disks and were subjected to a standard processing for optical microscopy, resulting in semiseriate sections 4 mm thick stained with hematoxylin and eosin. Descriptive and semiquantitative microscopic analysis of the tissue response around the disks was carried out by an experienced pathologist (AC; k test = 0.99) using an optical microscope. Quantitative analyses were carried out by a single researcher (SS; k test = 0.99). Semiquantitative analyses. To assess the degree of fibrosis in the reactive tissue around the disks, the number and density of collagen fibers 38 were classified according to the following scores: 0 = no fibrosis; 1 = mild fibrosis (individualized collagen fibers interspersed with negative spaces indicative of non-fibrous components of extracellular matrix); 2 = moderate fibrosis (individualized collagen fibers interspersed with areas of eosinophilic extracellular matrix without the typical linear and wavy arrangements); and 3 = intense fibrosis (non-individualized collagen fibers permeated by an eosinophilic extracellular matrix without the typical linear and wavy arrangements). To assess the intensity of the inflammatory infiltrate inthereactivetissuearoundthedisks,theconcentration of polymorphonuclear neutrophils was classified according to the following scores: 38 0 = no inflammatory infiltrate; 1 = mild inflammatory infiltrate (5 to 50 neutrophils in the reaction tissue); 2 = moderate inflammatory infiltrate (51 to 100 neutrophils in the reaction tissue); and 3 = intense inflammatory infiltrate ( >100 neutrophils in the reaction tissue). Quantitative analyses. Digital images of the histologic sections were analyzed with a software program. The total area (mm 2 ) and mean thickness (mm) at four diametrically opposed points on the reactive tissue around the disks were measured. Mean values were considered for comparisons among the groups. Statistical Analyses Analyses were performed using a statistical software program. iii Mann-Whitney U tests were used to compare the degree of fibrosis and severity of inflammatory infiltrate between smooth-surfaced and rough-surfaced disks. These parameters were based on average scores from 0 to 3. Kruskal-Wallis tests with Dunn post-tests were used to compare treatments and observation periods. Quantitative parameters (average area and thickness of the reaction tissue) were compared by two-way analysis of variance with a Tukey post-test. A significance level of 5% was adopted. ** Thera Lase, DMC Equipamentos, São Carlos, São Paulo, Brazil. Sigma-Aldrich, São Paulo, São Paulo, Brazil. Anasedan, Vetbrands, Jacareí, São Paulo, Brazil. Dopalen, Vetbrands. ii Free-Bac, EMBRAMAC, Itapira, São Paulo, Brazil. Ethicon, Johnson & Johnson, São Paulo, São Paulo, Brazil. ## Labina, Purina, Paulínia, São Paulo, Brazil. *** Synth, LabSynth Produtos para Laboratórios, Diadema, São Paulo, Brazil. Nikon Eclipse 80i, Nikon Instruments, Melville, NY. MicroPublisher 3.3 RTV, QImaging, Surrey, British Columbia, Canada. ImageJ 1.38x, National Institutes of Health, Bethesda, MD. iii Statistica, v. 10.0, StatSoft, Tulsa, OK. 643

4 Low-intensity Laser, apdt, and TBO Decontaminate Implant Surfaces RESULTS Of the 150 titanium disks implanted, six (two from group CS after day 84, two from group CR after day 84, one from group CR after day 28 and one from the rough-surfaced disks [group LILR] after day 28) were not found at the time of specimen collections. Semiquantitative Analysis No statistically significant difference in the degree of fibrosis (P = ) or severity of inflammatory infiltrate (P = ) was found between rough and smooth disks; therefore, the data were regrouped into five groups (negative control [NC], positive control [C], LIL, apdt, and TBO) of 10 specimens each, disregarding the surfaces (Figs. 2 and 3). Degree of fibrosis. In general, group C showed the lowest and group NC showed the highest degree of fibrosis of reactive tissue surrounding the disks. Figure 2. Microscopic views of the reactive connective tissue 7 days after the implantation of smooth-surfaced titanium disks in rats according to the experimental groups: A) NC; B) C; C) LIL; D) apdt; E) TBO. f = fibroblasts; c = collagen fibers; N = neutrophils; blue arrows = purulent exudate; MO = macrophages; red arrow = fibrinous exudate; V = blood vessels. White spaces correspond to the former locations of the disks. (Hematoxylin and eosin; original magnification 40.) Black arrows refer to macrophages. 644 Volume 84 Number 5 However, when compared pairwise, a significant difference was detected between only group C and group NC (P = ). Regarding observation periods, the degree of fibrosis after day 7 was lower than after days 28 and 84 (P = ). There was no significant difference between the findings after day 28 (P = ) and those observed after day 84 (P = ; Fig. 4). Severity of the inflammatory infiltrate. When all groups were considered together, there was a significant difference in the severity of the inflammatory infiltrate between day 7 and the other observation times (P = ). After day 7, group NC showed the lowest score, without a significant difference among the groups for any of the periods studied (P = ; Fig. 5). Quantitative Analysis Data obtained from the measurements of area and thickness of the reactive tissue surrounding the disks are presented in Table 1. The area of reactive tissue surrounding roughsurfaced disks ( mm2) was larger than Figure 3. Microscopic views of the reactive connective tissue 7 days after the implantation of rough-surfaced titanium disks in rats for the following experimental groups: A) NC; B) C; C) LIL; D) apdt; E) TBO. N = neutrophils; blue arrows = purulent exudate; MO = macrophages; red arrow = fibrinous exudate. White spaces correspond to the former locations of the disks. (Hematoxylin and eosin; original magnification 40.) Black arrows refer to macrophages.

5 Salmeron, Rezende, Consolaro, et al. Figure 4. Degree of fibrosis in rat subcutaneous reactive tissue around titanium disks treated with TBO, apdt, and LIL and in the C and NC groups. Figure 5. Severity of inflammatory infiltrate in rat subcutaneous reactive tissue around titanium disks treated with TBO, apdt, and LIL and in the C and NC groups. that surrounding smooth disks ( mm 2 ; P = ). The group NC disks (both smooth and rough) showed a significantly lower mean area of reactive tissue than the other disks after day 7 (P = ) and a lower mean area than group C disks (P = ) after day 84. The area of reactive tissue in group NC showed no significant change throughout the study (P >0.9992). Group C showed the largest area of reactive tissue after day 7, without a significant difference compared with group LIL (P = ) and group TBO (P = ). Only group apdt showed a smaller area than group C (P = ), but this was still larger than group NC (P = ). After day 28, areas of reaction tissue of all treatments groups were not statistically different (P >0.9684). The only exception was group C after day 84, which did not show a uniform pattern for any of the parameters tested. Because, overall, rough-surfaced implants showed larger areas of reactive tissue than smooth implants, separate comparisons with rough-surfaced disks were carried out. No treatment showed statistically different results on rough-surfaced implants after day 7 (P = 1), but all of them produced larger areas than sterile disks. After days 28 and 84, there was no difference in the reactive tissue area among the groups (P >0.1043). There was no significant difference in the thickness of reactive tissue between rough and smooth disks (P = ). Group NC showed significantly thinner reactive tissue than the other groups after day 7(P = ), but group C was not significantly different from all the treatment groups (P >0.4752) at the same observation time. In group LIL, tissue was thicker after day 7 than after days 28 and 84 (P = ). At the day 28 and day 84 observation times, there was no significant difference among the groups, including group NC (P >0.1822). Groups apdt and TBO showed this same behavior. DISCUSSION The results of this study demonstrate that LIL, apdt, and TBO are efficient at decontaminating titanium surfaces inasmuch as the degree of fibrosis and severity of inflammatory infiltrate for these treatments are similar to those observed for sterile surfaces. No single decontamination method could be considered superior to any other because after day 28 all of the disks produced a similar tissue reaction. Statistically significant differences were seen up to day 7 of healing and were greater when sterile implants and untreated contaminated implants were compared. Not even the implant surface texture had a significant influence on the response of tissue to treatments, with the exception of the reactive tissue area, which was larger in the rough-surfaced implants. These observations do not agree with the conventional wisdom that rough surfaces offer microorganisms better protection from removal during oral hygiene procedures 7 and that the bond between LPSs and metallic surfaces is extremely strong. 3,4 LIL, apdt, and TBO were chosen for decontaminating titanium surfaces because they are proven methods that result in benefits in the treatment of peri-implantitis. 36 The literature is rich in publications that attest to the viability of the treatment of peri-implantitis with different types of LIL, 6,21,22,24-27 apdt, and TBO 32,35 with various photosensitizing agents and isolated dyes that, when fixed to the bacterial cell wall, produce cytotoxic effects. Phenothiazine dyes have intense absorption in the 620- to 660-nm range and are useful in apdt because they are within the therapeutic window required not only for the efficient penetration of light in tissue, but also for the sufficient production of singlet oxygen. 39 Studies have shown that TBO is an effective photosensitizer to various bacteria, including species found in the oral cavity. 23,33,34 A study conducted in humans by Dörtbudak et al. showed that photosensitization with TBO plus irradiation with a diode laser (690 nm) for 1 minute resulted in a significant bacterial reduction on 645

6 Low-intensity Laser, apdt, and TBO Decontaminate Implant Surfaces Volume 84 Number 5 Table 1. Mean Area and Thickness of Reactive Tissue Following the Implantation of Titanium Disks (N = 144) in Rat Subcutaneous Tissue Area (µm 2 ) 7 Days 28 Days 84 Days Smooth Rough Smooth Rough Smooth Rough NC ⁶ Aa ⁶ Aa ⁶ Aa ⁶ Aa ⁶ Aa ⁶ Aa C ⁶ Ab ⁶ ABb ⁶ Ba ⁶ Aba ABb ⁶ Aba LIL ⁶ Acd ⁶ Abc ⁶ Ba ⁶ Ba x 10⁶ Ba ⁶ Ba apdt ⁶ Aade ⁶ Bc ⁶ Ca ⁶ Ca ⁶ Ca ⁶ Ca TBO ⁶ Ace ⁶ Abc ⁶ Ba ⁶ Ba ⁶ Ba ⁶ Ba Thickness (µm) 7 days 28 days 84 days Smooth Rough Smooth Rough Smooth Rough NC Aa Aa Aa Aa Aab Aa C Ab Ab Ba Ba Ba Ba LIL Abc Abc Ba Ba Bb Ba apdt Ac Ac Ba Ba Bab Ba TBO Ac Abc Ba Ba Bb Ba Different lowercase letters in the same row and different uppercase letters in the same column indicate statistically different values (P 0.05). implant surfaces showing clinical and radiographic signs of peri-implantitis. 23 The authors interestingly observed that the application of TBO alone resulted in a significant reduction in Prevotella intermedia and Aggregatibacter actinomycetemcomitans, but not in Porphyromonas gingivalis, comparedwithinitial bacterial counts. This finding could not be attributed to a greater susceptibility to the dye from black-pigmented bacteria, but it was suggested that a variable bonding behavior of the dye to the different bacterial membranes may occur. Then, the photosensitization provided by the TBOcauseddamagetothe bacterial membrane when the dye was activated by the laser. Their results are consistent with the histologic findings of the current study and, at least partially, could explain the apparent superiority of apdt to TBO alone. As expected, semiquantitative analysis revealed moderate inflammation without abscess formation for sterile disks after day 7. These disks produced a similar tissue reaction after days 28 and 84, with the formation of a capsule that was gradually more collagenized and infiltrated to a lesser degree by neutrophils, indicating a resolution of the acute inflammation over time. By contrast, after day 7, the untreated contaminated disks produced purulent inflammatory infiltrate in a large portion of the tissue. This result probably caused the expelling of the five disks from group C that were not located during the biopsies. After days 28 and 84, however, the intense inflammatory reaction observed in group C gave way 646

7 Salmeron, Rezende, Consolaro, et al. to a fine reactive tissue that was comparable to that seen for sterile disks at the same observation times. LIL-treated disks showed similar effects at the three observation times. After day 7, most of the formed abscesses were less intense than those seen in group C disks (one disk in the LIL R group [day 28] was also probably expelled by an abscess). After days 28 and 84, the LIL-treated disks showed behavior similar to disks from the other treatment groups. After day 7, group apdt behaved similarly to groups C, LIL, and TBO. However, the quantity of accumulated neutrophils and purulent exudate was much smaller in group apdt than in the others. This difference also disappeared after days 28 and 84. These data agree with other studies 23,34,40 that reported apdt is very efficient at reducing microbes on titanium surfaces with different textures, compared with isolated treatments with LIL and TBO. The findings of the present study also corroborate reports of no significant difference among the effects of apdt, conventional flap therapy, and irrigation with chlorhexidine. 31 Indeed, surface texture did not affect the degree of fibrosis or the severity of the inflammatory infiltrate. As these two parameters are proportionally related to the intensity of the inflammatory reaction, it may be that the topographic differences between the surfaces of the disks used in this study were not large enough to produce differences in soft tissue behavior following implantation. The treatments used did not cause damage to the surrounding tissues, thereby fulfilling one of the stated goals of treatment methods for peri-implantitis: maintaining the original integrity of surrounding tissues. 18,19 Nakazato et al. 8 noted that surface texture interfered with bacterial adhesion during the first 48 hours from the beginning of the natural deposition of bacterial biofilm but did not have any qualitative effects after that time. They attributed this finding to differences in surface free energy between materials with different textures, so that smooth implants (with low surface energy) retain more cocci than roughsurfaced implants (with high surface energy), which retain more spirochetes and mobile microorganisms. 11,41,42 This could provide support for the hypothesis that the similar tissue responses observed among treatments were the result of similar composition of the microbial biofilm on both types of surfaces of the titanium disks. In all groups, the degree of tissue fibrosis was lower at day 7 than at the other observation times. In general, there was a difference between group C and group NC only. This may be explained by the greater level of contamination on the untreated contaminated disks in relation to the others, which probably caused a more intense inflammatory response. However, when the degree of fibrosis seen in group C was compared to that seen in treatment groups, no statistical difference was observed. The same can be said of the severity of the inflammatory infiltrate, which was greater after day 7 than at the later times, with no difference among groups (although the most common score for sterile disks [2] was lower than that for the other groups [3]). After day 28, all groups showed similar levels of severity of the inflammatory infiltrate. Assessments of the area and thickness of reactive tissue surrounding implanted materials have been used to assess the biocompatibility of these materials. 38 More intense inflammation involves a larger number of cells and a greater volume of interstitial fluid, resulting in larger and thicker reactive tissue. Rough-surfaced disks showed larger areas of reactive tissue than smooth disks. Inasmuch as the rough surface is theoretically more difficult to decontaminate, the observed difference was attributed to the texture. For this reason, rough-surfaced implants were analyzed separately, aiming at determining if one of the treatments was more effective than the others. The absence of such superiority in this study corroborates the findings of Parlar et al., who found that saline solution alone plus autoclaving were successful at cleaning and decontaminating implants. 43 Sterile disks showed smaller areas of reactive tissue than the other disks at days 7 and 28. The greatest area was observed in untreated contaminated disks at day 7, without significant difference compared with LIL and TBO at the same time (P = and P = , respectively). One interesting finding was that the only group with a smaller area than group C at day 7 (but still bigger than the NC group) was group apdt, which suggests that this treatment was slightly superior to the others. Reactive tissue thickness was calculated with the aim of complementing the area results because both parameters reflect the same phenomenon. However, smooth and rough disks showed no significant difference in mean reactive tissue thickness (P = ). This was attributed to the standardization of the tissue regions measured, which did not always match the thickest regions in each specimen; despite this, the results showed that sterile disks produced thinner tissue and untreated contaminated disks produced thicker tissue than the other disks at all the observation times. The fact that all groups showed similar reactive tissue area and thickness after day 28 suggests that contaminated implants (even untreated ones) placed in living tissue with no access to the external environment may eventually be well tolerated by the body. Taken together, the presented results suggest that the method by which titanium surfaces are 647

8 Low-intensity Laser, apdt, and TBO Decontaminate Implant Surfaces Volume 84 Number 5 decontaminated may interfere to some extent with tissue behavior during the initial phases of tissue repair and that apdt may be slightly superior in this respect to isolated LIL and TBO. This information is especially important when considering a protocol for treating peri-implantitis because it is in the first days of the tissue repair process that cells coming from the epithelial, connective, and bone tissue compete for colonizing the surgical wound. In clinical conditions, epithelial cells would be the first to reach the metallic surface over 7 to 15 days. 44 It appears that during this period the decontamination methods must show differences if they are to be useful in treating peri-implantitis. If the behavior shown by apdt in this study can be duplicated in the actual peri-implant environment, it is possible that bone precursor cells may be capable of adhering to treated surfaces from which epithelial cells are excluded, thereby favoring reosseointegration. On the other hand, it is noteworthy that all the methods studied have an important limitation when applied in the clinical setting, which is the presence of bleeding. Red blood can absorb a large portion of energy produced by lasers from the red band (<620 nm or >740 nm). In the case of a red laser, as the one used in this study, blood would not compete with TBO, but it could act as a physical barrier for the dye to reach the bacterial membrane. Moreover, excessive bleeding may prevent the laser from reaching deeper areas of bone defects, thereby reducing its effectiveness. 23,27 If this is the case, a good control of transsurgical bleeding during apdt therapy would be recommended. CONCLUSIONS Given the limitations of this experiment, LIL, TBO, and apdt were equally efficient at decontaminating smooth- and rough-surfaced titanium implants, based on the response of the subcutaneous connective tissue of the rats and that apdt performed slightly better after day 7. The texture of implants did not appear to affect the intensity of the inflammatory response induced by its presence nor the decontamination achieved by the studied methods. ACKNOWLEDGMENTS This study was made possible by the financial support of The State of São Paulo Research Support Foundation, São Paulo, São Paulo, Brazil to Dr. Rezende (FAPESP, grant 2010/ ) and by the post-graduate program in Applied Dental Sciences of the Bauru School of Dentistry, University of São Paulo. The authors thank the professional science editor and the native English-speaking copy editor for their reviews. The authors report no conflicts of interest related to this study. REFERENCES 1. 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J Clin Periodontol 1982;9: Correspondence: Msc. Samira Salmeron, Alameda Octávio Pinheiro Brisolla, 9-75/Vila Universitária, Bauru, São Paulo, Brazil Fax: ; s.salmeron@usp.br Submitted March 12, 2012; accepted for publication May 13,