Photodiagnosis and Photodynamic Therapy (2015) 12, 575—580
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Effects of antimicrobial photodynamic therapy and surgical endodontic treatment on the bacterial load reduction and periapical lesion healing. Three years follow up Aguinaldo S. Garcez Ph.D. a,∗, Julio G. Arantes-Neto b, Debora P. Sellera b, Eduardo Rodrigues Fregnani c a
São Leopoldo Mandic Dental Research Center, Campinas, SP, Brazil Associac¸ão Paulista de Cirurgiões Dentista, São Paulo, SP, Brazil c Hospital Sírio-Libanês, São Paulo, SP, Brazil b
Received 3 March 2015; received in revised form 12 May 2015; accepted 1 June 2015 Available online 10 June 2015
KEYWORDS Endodontics; Antimicrobial; PDT; Periapical surgery; Bone healing
Summary Besides the advances in endodontics, there are situations in which surgery is necessary to retain a tooth that otherwise would be extracted. This study analyzes the microbial reduction after conventional periapical surgery followed by antimicrobial photodynamic therapy (aPDT) in 3 years follows up. Twenty-eight teeth needing periapical surgery were enrolled in this study. Microbiological samples were taken after: (1) accessing the lesion, (2) surgical procedure, and (3) aPDT. The teeth received a full mucoperiosteal flap, osteotomy with a high-speed bur, manual curettage of lesion and of the root, root-end resection and retrograde cavities prepared using ultrasonic retro-tips. After the conventional procedure the cavities received an aqueous solution of methylene blue (60 M, 3 min) and were irradiated with a diode laser = 660 nm (6 min, 15 J). After aPDT, a retrograde filling with mineral trioxide aggregate, flap repositioning, and sutures conventionally ended the procedure. In all the cases, a periapical X-ray exam was taken before and after the surgical procedure. The microbiological samples showed an overall significant reduction, surgical procedure achieved a mean reduction of about 3.5 log while aPDT achieved a 5 log reduction. After 36 months of follow up the patients had, in average a reduction of 78% of the periapical lesion area. It appears that surgical endodontic
∗
Corresponding author at: Rua Taquara Branca, 78 ap 36A, 02069-000 São Paulo, SP, Brazil. E-mail addresses:
[email protected] (A.S. Garcez),
[email protected] (J.G. Arantes-Neto),
[email protected] (D.P. Sellera),
[email protected] (E.R. Fregnani). http://dx.doi.org/10.1016/j.pdpdt.2015.06.002 1572-1000/© 2015 Elsevier B.V. All rights reserved.
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A.S. Garcez et al. treatment associated with antimicrobial photodynamic therapy highly improves the microbial reduction compared to the traditional technique and this could directly affect the treatment prognosis and periapical lesion healing. © 2015 Elsevier B.V. All rights reserved.
Introduction Besides the advances in endodontic treatment [1], there are situations in which surgery are necessary to retain a tooth that otherwise would be extracted [2]. Endodontic surgical treatment prevents or minimize the invasion of bacteria and their by-products from the root canal into periradicular tissues [3] and indications for periradicular surgery is the removal of non reachable bacteria with conventional endodontic treatment, due to complex root canal anatomy, procedural accidents, irretrievable materials in the root canal, symptomatic cases, horizontal apical fracture, biopsy and corrective surgery [4,5]. Antimicrobial photodynamic therapy (aPDT) has been studied [6,7] as a promising approach to eradicate oral pathogenic bacteria that cause endodontic diseases [8], periodontitis [9], periimplantitis [10] and caries [11—13]. The combination of endodontic treatment and aPDT has been shown as an effective approach in reducing bacterial load in in vitro and in vivo models [14—19]. However, in the literature there is no information about the effect of aPDT associated with conventional endodontic surgery regarded to microbial reduction and even more if the noxious components produced on the periapical tissue could slow down the healing process. This study evaluates the long-term effectiveness of aPDT and conventional endodontic surgery in reducing the bacterial load and its effects on bone healing in cases of large periapical lesion.
Materials and methods Twenty-eight teeth were enrolled in this study from 22 patients with periapical lesions who had been previously treated with endodontic treatment. All the patients were in good health, checked by individual anamnesis interview, and between the ages of 17 and 52 years. All the teeth (incisives, canines and premolars) presented signs and symptoms of periapical periodontitis and apical bone lesion detected by radiography. The protocol was reviewed and approved by the Institutional Review Board of the São Paulo University. A full mucoperiosteal flap, osteotomy, manual curettage (periapical lesion and external root surface), root-end resection, and retrograde cavities preparation using ultrasonic retro-tip were performed as a conventional treatment (Fig. 1). After the conventional procedure, the cavities received an aqueous solution of methylene blue (60 M — 3 min of preirradiation time) and a diode red laser ( = 660 nm, P = 40 mW for 3 min, E = 7.2J). The cavity was then dried and the tip of the laser was changed to allow access to the retrograde cavity. The irradiation inside the retrograde cavity was performed with an optical fiber (˚ = 200-mm, MMOptics, São Paulo, Brazil) [20].
Subsequently to aPDT, a retrograde filling with mineral trioxide aggregate (MTA Angelus — Londrina, Paraná, Brazil), flap re-positioning, and sutures ended the procedure. Microbiological samples, using sterile swabs, were taken, after osteotomy and accessing the bone lesion, after surgical procedure and after aPDT. The samples were removed from the anaerobic transport medium (VMGA III), placed in BHI broth, and vortexed for 30 s. Serial dilution and colony-forming units (CFU) enumeration were performed [21] after incubation, on BHI agar plates, inside a microaerophilic chamber with 5% oxygen, 15% carbon dioxide, and 80% nitrogen for 72 h at 37 ◦ C. At each stage of the treatment (initial, after endodontic surgery, and after aPDT), CFUs were counted. Survival fractions were calculated from each patient taking into account its initial bacterial load [14]. The method of culture was selected to assess the microbial load usually found in infected root canals [22]. However, no attempt was made to identify the specific microbial flora during the process. A periapical radiograph, with an individual positioning support, was taken before and after the treatment and after 36 month to evaluate the decrease of the periapical lesion area using Image J (National Institute of Health, USA) software.
Statistical analysis Statistical comparisons between means were performed with a paired t test using Microsoft Excel; 2-tailed p values are reported.
Results The microbiological samples confirmed the presence of infection in all teeth. The initial infectious burden varied widely between patients. This variation was probably caused by differences of the individual root anatomy, duration of the infections and dimension of lesion (Fig. 2). After conventional therapy, the mean infectious burden was reduced to approximately 3.5 log. The mean infectious reduction after subsequent aPDT was around 1.5 log. The overall mean log reduction after the combined therapies was 5 logs and this was significantly greater than that achieved by endodontic surgery alone (p < .0005). None of the lesion treated had 100% microbial reduction after surgical treatment, whereas in 8 lesions were not possible to detect bacteria after combinations of endodontic treatment and aPDT. Radiographic follow-up showed a decrease of the lesion area for all teeth. The decrease of the lesion area did also vary widely between individual teeth, probably for the same reasons the contamination vary and also due the individual
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Figure 1 Conventional treatment sequence. (A) Initial case — fistulae on central incisor. (B) Bone lesion after mucoperiosteal flap. (C) Retrograde cavities preparation using ultrasonic retro-tips. (D) Aspect of the lesion after surgery procedure. (E) Methylene blue application. (F) Irradiation of the retro cavity with the laser coupled to the 200 m optical fiber. (G) Irradiation of the lesion with the laser tip. (H) Retrograde filling with mineral trioxide aggregate. (I) Sutures.
variation on healing for each patient. The area reduction (Fig. 3) was, in average, 78% (65—100%).
Discussion Antimicrobial PDT was highly effective in reducing bacterial load in an ex vivo model, in patients and even in
Figure 2
a drug-resistant endodontic infection [14,15,19,20]. This study shows for the first time, in vivo, the combination of surgical endodontic treatment and aPDT for antimicrobial reduction in periapical lesions. When obturation of endodontic treatment is performed in a positive culture, the success rate is reduced and retreatment is indicated [23,24]. In some situations surgery
Microbial reduction on each stage of the treatment.
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Figure 3 Representative radiographic images of one case. (A) Initial periapical lesion; (B) Measurement of the lesion area before surgery; (C and D) immediately after surgery and aPDT; (D and F) Follow up after 3 years.
is necessary to retain a tooth that otherwise would be extracted due to insufficient decontamination [2]. Reduction of the microorganisms, in case of periapical lesions, has been a challenge, due to polymicrobial contamination [26,27]. Mechanical instrumentation alone, during the surgery process, may not be able to completely eradicate contamination [28,29]. Therefore, chemical disinfection is necessary to optimally decontaminate the lesion [30]. Various medicaments have been widely advocated to help eliminate bacteria, reduce periapical inflammation, and induce healing [31]. Antibiotics have been used [25] and among them a mixture of ciprofloxacin, metronidazole, and minocycline has been shown to be very effective in vitro and in situ, especially in large cyst-like periradicular lesions [31,32]. Another advantage of aPDT is it strict local activity, when compared to antibiotics, avoiding damage to internal flora, such as intestinal and oral normal microflora. In conventional treatment, reducing the bacterial count is accomplished by a combination of mechanical therapy, and antimicrobial medicaments [33]. But the long-term use of antimicrobial agents can be rendered ineffective by resistance developing in the target organisms [25]. Culturing of root canal microflora is complicated, and it demands microbiological facilities in close proximity to the dental office to ensure that microorganisms do not die in
transit. However, it is the most effective short-term means of evaluating the disinfection of root canals in vivo [8,23]. To avoid this problem, canal samples were cultured within 1 h after the sample had been taken, following a previous methodology used by our group [14]. It was decided that a quantitative method to count the total microorganisms assessed inside the root canal would be appropriate because the aim of this study was to verify the number of microorganisms present after endodontic surgery and subsequent antimicrobial PDT. Antimicrobial PDT produces additional decontamination without creates microbial resistance. In addition, the literature has showed that is safety to use aPDT against microorganisms near normal cells [34,35]. Cytotoxicity seams to be significantly less in aPDT compared with conventional antimicrobial irrigation, since the effects of chemical irrigants on periapical tissues are well known [36]. In vitro, aPDT produced 97.7% of E. faecalis killing and only 30% fibroblast dysfunction [34]. Also Xu et al. [37] suggested that there is a safe therapeutic window whereby aPDT can inactivate endodontic pathogens without affecting host cell viability. Even more, Qiao et al. [38] found that aPDT exhibited no cytotoxicity to human periodontal cells. The microbial reduction achieved in this study showed a superior result when compared to results of studies in root canal when aPDT is associated with endodontic conventional
Effects of antimicrobial photodynamic therapy and surgical endodontic treatment treatment [16,39]. It is possible that the access by light and by the photosensitizer in the lesion is better than in the root canal (mainly by the root anatomy and internal volume), moreover the use of the optical fiber enhanced decontamination. In addition, the surrounding tissue could promote light back scattering thus increasing the number of photons available to photoreaction. Thirty-six months after the treatments all the patients were asymptomatic, all the teeth had returned to then normal function and radiographic exams showed a reduction of periapical lesion area. The decrease of the lesion area indicates the success of the treatment and the amount of noxious compound produced by aPDT, in the parameters used in this study, clearly is not harmful to the surrounding tissue, even achieving a significative microbial reduction, as proposed before [34,35]. Besides the limitation of periapical radiography in precisely detect reduction of a volumetric lesion, Liang et al. [40] have shown that when comparing Cone Bean Computer Tomography (CBCT) and Periapical Radiography (PA) in Radiographic healing after a root canal treatment, CBCT detected significantly more post-treatment lesions than PA (P = .038), but the percentages of absence and reduction of the radiolucency together revealed by CBCT and PA were similar (P = .383). A previous study, of our group, compared the periapical lesion healing after conventional endodontic treatment associated with aPDT [41]. The decrease of the lesion area in the previous study was 86% and was higher than in this study, which achieved 78% of lesion reduction. Bone curettage, root resection and retro-obturation, during surgery procedures could justify the lowed healing rate, since this procedures could promote additional trauma to the area compared to conventional endodontic treatment. In conclusion, our results suggest that the use of aPDT as an adjuvant to conventional endodontic surgery leads to a significant further reduction of bacterial load. Antimicrobial PDT offers an alternative method of destroying microorganisms after using conventional therapy.
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