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BACKGROUND: The cytotoxic effect of chemotherapeutic agents to the oral mucosa, as a side-effect of cancer treatment, is a major problem. Cooling the oral mucosa using ice chips in conjunction with chemotherapy is known to reduce the severity of oral mucositis. However, although the use of ice chips is of clinical value, this method of cooling has inherent problems including discomfort for the patient, non-uniformity and fluctuations in cooling temperature throughout the oral cavity. Furthermore, despite being used clinically, it is not known what reduction in temperature is required to prevent oral mucositis. The aim of the present study was therefore to determine in vitro if the cytotoxic effect of 5-Fluorouracil (5-FU) on the oral mucosa could be reduced by lowering the temperature during chemotherapeutic treatment. METHODS: Tissue engineered oral mucosal (TEOM) models were incubated at 20, 25, 30, or 35 °C for 30 minutes followed by exposure to a clinically relevant concentration of 5-FU (162 μg/ml) for two hours and compared to untreated models (35°C). Cell viability and inflammatory cytokine production (IL-6 and TNF-α) were measured using PrestoBlue®and ELISA, respectively. RESULTS: TEOM models incubated at 20°C showed an increased cell viability and had a reduced IL-6 and TNF-α production compared to models treated with 5-FU incubated at 35°C. CONCLUSION: This study demonstrates a reduced cytotoxic effect to the TEOM by reducing the temperature of the tissue during chemotherapy treatment and suggests that decreasing the temperature to 20°C could have clinical advantages. This article is protected by copyright. All rights reserved.

The generation of tissue-engineered epithelial models is often hampered by the limited proliferative capacity of primary epithelial cells. This study aimed to isolate normal tonsillar keratinocytes (NTK) from human tonsils, increase the lifespan of these cells using the Rho kinase inhibitor Y-27632 and to develop tissue-engineered equivalents of healthy and infected tonsil epithelium. The proliferation rate of isolated NTK and expression of c-MYC and p16INK4A were measured in the absence or presence of the inhibitor. Y-27632-treated NTK were used to generate tissue-engineered tonsil epithelium equivalents using de-epithelialized dermis that were then incubated with Streptococcus pyogenes to model bacterial tonsillitis, and the expression of pro-inflammatory cytokines was measured by cytokine array and ELISA. NTK cultured in the absence of Y-27632 rapidly senesced whereas cells cultured in the presence of this inhibitor proliferated for over 30 population doublings without changing their phenotype. Y-27632-treated NTK produced a multi-layered differentiated epithelium that histologically resembled normal tonsillar surface epithelium and responded to S. pyogenes infection by increased expression of pro-inflammatory cytokines including CXCL5 and IL-6. NTK can be isolated and successfully cultured in vitro with Y-27632 leading to a markedly prolonged lifespan without any deleterious consequences to the cell morphology. This functional tissue-engineered equivalent of tonsil epithelium will provide a valuable tool for studying tonsil biology and host-pathogen interactions in a more physiologically relevant manner.