Quantitative analysis of radiation-induced DNA break repair in a cultured oral mucosal model

Oral mucositis is a major side effect of radiation therapy. Development of strategies for reduction of this problem calls for quantitative models. The goal of the present study was to test the feasibility of detecting double-strand breaks (DSBs) and DSB repair proteins upon radiation of mucosa in a 3-dimensional culture system using morphology and immunohistochemistry. Human oral keratinocytes and fibroblasts were seeded onto and into an acellular dermal carrier to produce a cultured mucosal substitute (CMS). CMSs were gamma-irradiated with 0, 2, and 12 Gy. One group received 4 Gy through 2 Gy fractions with a 24-h interval. Radiation-induced damage was quantified using hematoxylin and eosin (H&E). DSBs and DSB repair proteins were visualized and quantified using antibodies against P53 binding protein 1 (53BP1), MRE11, and RAD51. As in cell culture, CMSs showed intranuclear loci of damage and repair, mostly in the proliferative basal cell layers. Maximum percentages of damaged basal layer keratinocytes were 54.8% using H&E (12 Gy) up to 78.9% (12 Gy) for 53BP1. This study shows the feasibility of DNA repair markers to quantify radiation damage. This is an important step forward in the study of mucositis and the development of treatment and prevention strategies, proving once more the power and clinical importance of tissue engineering.