The effects of ionizing radiation on dentin endogenous proteases

Author(s)

Sandra Ribeiro de Barros da Cunha

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Odontologia (FO/SDO)
• Defense date: 2019-01-18

Examining board members

Ana Cecilia Corrêa Aranha; Lorenzo Breschi; Ana Karina Barbieri Bedran Russo

• Advisor: Ana Cecilia Corrêa Aranha

Abstract

Radiotherapy is one of the main treatments for head and neck cancer patients. Radiation-related caries and early restorations failures are side-effects with high rate of recurrence. As enzymatic degradation of collagen occurs mainly through the activity of matrix metalloproteinases (MMPs) and cysteine-cathepsins (CTs), the objective of this study was to evaluate the influence of in vivo and in vitro radiotherapy on endogenous proteases of the restored and non-restored dentin. In vivo irradiated teeth were extracted from patients who underwent clinical radiation protocols with a cumulative dose of radiation that ranged from 40 to 70 Gy. Extractions were performed 3 to 12 months after radiotherapy conclusion due to periodontal reasons. For the in vitro irradiated teeth, samples were submerged in distilled water with a total and single irradiation dose of 70 Gy. For gelatin zymography assay, irradiated in vivo, in vitro and non-irradiated groups were divided in two subgroups: 1) mineralized or 2) demineralized with 10% phosphoric acid. Dentin proteins were extracted and submitted to zymographic analysis in accordance to Mazzoni et al., 2007. For in situ zymography, specimens were divided into 6 groups, according to its irradiation form (non-irradiated and irradiated in vitro) and the adhesive system tested (Adper Single Bond, 3M ESPE, ClearFil SE Bond, Kuraray or Scotchbond Universal, 3M ESPE) using a self-quenched fluorescein-conjugated gelatin as the endogenous proteases substrate. The endogenous gelatinolytic enzyme activity was assessed by confocal laser-scanning microscope (Zeiss LSM 780-NLO, Carl Zeiss Microscopy GmbH). For SEM analysis of the HL, restored specimens were submitted to a pre-embedding immunolabeling technique using primary monoclonal antibody anti-CT-K and anti-CTB and a secondary antibody conjugated with 15nm gold nanoparticles. Radiotherapy groups presented increased gelatinolytic activity on both restored and non-restored dentin. MMP-2 and MMP-9 active form presented higher expression on both irradiated groups for non-restored dentin. Labeling for CT-K and CT-B did not differ from irradiated to non-irradiated groups. SE adhesives presenter weaker labeling for CT-K when compared to the E&R adhesive. Herewith, ionizing radiation may be able to influence the enzymatic activity of the endogenous proteins of restored and unrestored dentin (AU)

• FAPESP's process: 15/20297-3 - The effects of radiotherapy on the endogenous proteases of sound and restored dentin
• Grantee: Sandra Ribeiro de Barros da Cunha
• Support Opportunities: Scholarships in Brazil - Doctorate