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 8 Gy. 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 submocosal cellular components was a useful model. Keywordsradiation-induced oral mucositis; in vitro assay system; organotypic culture; pro-inflammatory cytokine Oral mucositis is a severe side effect for patients undergoing radiation and/or chemotherapy for head and neck tumors, clinically characterized by erythema, ulceration and pseudomembrane formation of the oral mucosa resulting in ulcers producing severe pain and Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 7,14,15,17 . These systems utilized an irradiated xenogeneic feeder layer of cells and/or medium that contained animal serum and pituitary extract. These cells and/or supplements add an unknown element to the culture conditions through cross-contamination with prions and/or slow viruses, and the culture medium for the cells is not chemically well defined. NIH Public AccessIn response to these limitations the authors developed a tissue-engineered three-dimensional (3D) human stratified oral mucosa, an ex vivo produced oral mucosa equivalent (EVPOME) in a culture system devoid of animal serum products, pituitary extracts and a xenogeneic irradiated cell feeder layer 10 . EVPOME can be utilized as a more accurate representation of cellular behavior in situ to assess the effects of irradiation on cell behavior, cell...

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