Effect of zoledronate during the alveolar healing process in rats: study involving the major risk factors for developing osteonecrosis of the jaws and assessment of antimicrobial photodynamic therapy (aPDT) as preventive therapy

Abstract

Bisphosphonates (BPs) are a class of drugs used in the treatment of some bone diseases or osteotropic malignancies. BPs exert potent inhibitory effect on bone resorption. Among the adverse effects is the occurrence of bisphosphonate-related osteonecrosis of the jaws (BRONJ). The pathogenesis of the disease is poorly understood, which hinders its prevention and treatment. Among the risk factors for the onset of BRONJ are: chronic use of nitrogen-containing-BPs, especially zoledronate, intravenous route of administration, female gender, older age and oral surgeries, especially exodontia. Among the few therapeutic approaches is preventive antibiotic therapy, which shows failure and adverse effects. Antimicrobial photodynamic therapy (aPDT) has antimicrobial properties and potent biostimulatory effects, placing it as a potential therapeutic strategy for preventive BRONJ. The purpose of this study is to evaluate aPDT effect on alveolar socket and overlying mucosa of rats that show the main risk factors for the development of ONJ. One hundred and twenty rats (female; 20-month-old) will be divided into eight experimental groups: SAL, ZOL, SAL/FS, ZOL/FS, SAL/LLL, ZOL/LLL, SAL/aPDT and ZOL/aPDT. The duration of the treatment plan will be of seven weeks. Rats will receive intraperitonial injections of 0.45 ml solution of 0.9% sodium chloride (SAL, SAL/FS, SAL/LLL, SAL/aPDT groups) or 0.45 ml of same solution added with 100 mg/kg of zoledronate (ZOL, ZOL/FS, ZOL/LLL, ZOL/aPDT groups) with an interval of two days between the applications. After three weeks of treatment, all rats will be submitted to extraction of right mandibular first molar. In groups SAL/FS and ZOL/FS, wound will be covered with 1ml of methylene blue (photosensitizer) and maintained for 60s. In groups SAL/LLL and ZOL/LLL, rats will be submit to irradiation sessions of low-level laser therapy (LLL) at 0, 2 and 4 days after exodontia. In groups SAL/aPDT and ZOL/aPDT, rats will undergo sessions of aPDT at 0, 2 and 4 days after exodontia. After 28 postoperative days, all rats will be euthanized. Subsequently, the hemi-mandibles will undergo histological processing and staining by hematoxylin-eosin, picro-sirius red, Gram or will be submitted to immunohistochemistry. At the extraction site and mucosa overlying will be carried out: histopathological analysis, histomicrobiological analysis for the detection of bacteria, histochemical analysis of collagen maturation level, histometric analysis of new bone tissue and immunohistochemical analysis directed to assess the following biomarkers: proliferating cell nuclear antigen (PCNA), BCL-2 associated X protein (BAX), cleaved caspase-3, platelet/endothelial cell adhesion molecule-1 (PECAM-1), vascular endothelial growth factor (VEGF), runt-related transcription factor-2 (RUNX-2), osteocalcin (OCN), tartrate-resistant acid phosphatase (TRAP), receptor activator of nuclear factor- k B (RANKL), osteoprotegerin (OPG), bone morphogenetic protein 2/4 (BMP 2/4) and esclerostin. Results will be analyzed under light microscopy and will be submitted to quantication and statistical analysis at 5% significance level. (AU)