Development of an in vitro model for radiation-induced effects on oral keratinocytes

Abstract 

Changes in epithelial cell activity and the production of pro-inflammatory cytokines were examined utilizing an organotypic culture system as an in vitro model to study the effects of radiation on oral keratinocytes to simulate what is thought to occur in radiation-induced oral mucositis. Monolayer cultures of oral keratinocyte were irradiated by varying the dose. Cell injury was assessed using a colony forming efficiency (CFE) assay. Third passage oral keratinocytes were seeded onto AlloDerm^® to form a 3D construct of an ex vivo produced oral mucosa equivalent (EVPOME) which was irradiated with 0, 1, 3 and 8Gy. Formalin-fixed sections of the EVPOME were used for histology and immunohistochemistry to examine proliferative capacity. Epithelial cell viability of EVPOME was measured by MTT assay. Spent culture medium was used to determine post-radiation pro-inflammatory cytokine production. Basal cells became more swollen and pyknotic as radiation increased, implying loss of cell viability also determined by MTT assay. The number of Ki-67 immunopositive cells and CFE showed negative correlation with radiation, indicating loss of cell proliferative capacity. The production of pro-inflammatory cytokines, IL-1α and IL-8, tended to increase in a radiation dose dependent manner. The EVPOME lacking submucosal cellular components was a useful model.

Key words: radiation-induced oral mucositis, in vitro assay system, organotypic culture, pro-inflammatory cytokine