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 submucosal cellular components was a useful model.
Oral mucositis is a severe side effect for patients undergoing radiation and/or chemotherapy for head and neck tumours, clinically characterized by erythema, ulceration and pseudomembrane formation of the oral mucosa resulting in ulcers producing severe pain and discomfort that make dietary intake difficult . The severity of the signs and symptoms may increase patient morbidity and culminate in the cessation of cancer therapy treatment .
Monolayer cultures of oral keratinocytes are often used to assess the effects of ionizing radiation, but are two dimensional and do not represent accurately what is occurring in situ where the cells live in three dimensions . Several investigators have utilized artificial reconstructed skin or mucosa substitutes for clinical or basic science studies to evaluate the functional aspects of mucosa and the cytotoxic effects to radiation . 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.
In 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 . EVPOME can be utilized as a more accurate representation of cellular behaviour in situ to assess the effects of irradiation on cell behaviour, cell kinetics and the release of pro-inflammatory cytokines.
Recent studies elucidated more complex mechanisms of oral mucositis in which dynamic multiple molecular and cellular events occur, the activation of transcription factors such as nuclear factor-κB (NF-κB) and the upregulation of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) . Five biological stages of oral mucositis were defined: initiation, primary damage response, signalling amplification, ulceration and healing . The present study was carried out to assess the effect of ionizing radiation on oral mucosa keratinocytes without submucosal cellular components and to examine the metabolisms of oral mucosa keratinocytes within the EVPOME during the early stages of development of oral mucositis. This was accomplished by exposing the EVPOME model to gradually increasing doses of radiation. Cell viability was then assessed using MTT assay for cellular metabolism . Cell proliferation was determined by quantifying the presence and expression of a cell proliferation antigen . The release of pro-inflammatory cytokines, IL-1α and IL-8, was quantified in the culture medium before and after irradiation, as they are thought to effect the primary damage reaction/signalling amplification caused by ionizing radiation .
Materials and methods
Monolayer culture of oral mucosa keratinocyte
Oral mucosa keratinocytes were obtained from surgically discarded oral mucosa (six samples: two male and four female). Sample tissues were washed three times in Dulbecco’s phosphate-buffer saline (PBS) (BD Whittaker, Walkersville, MD, USA) supplemented with 15 μg/ml gentamycin and 7.5 μg/ml fungizone (GIBCO/Invitrogen, Carlsbad, CA, USA). After trimming subcutaneous tissue and blood, samples were digested in 0.04% trypsin (Sigma, St. Louis, MO, USA) overnight at room temperature. 0.0125% trypsin inhibitor (Cascade Biologics, Portland, OR, USA) was used to neutralize the trypsin and the epidermal side of the mucosa was gently scraped with a scalpel to detach the mucosa keratinocytes. The cell suspension was filtered through a 240 μm nylon mesh, centrifuged at 1100 rpm for 5 min, resuspended in chemically defined serum-free medium (EpiLife ® , Cascade Biologics) supplemented with 0.06 mM calcium concentration, 12.7 mg/ml gentamycin and 192 μg/ml fungizone (GIBCO/Invitrogen), and then seeded into flasks at an initial density of 1.0 × 10 5 cells/cm 2 . Cells were incubated at 37 °C, 5% CO 2 in humidified air. Medium was changed every other day. Once keratinocyte cultures reached 60–70% confluency, they were split and passaged with 0.025% trypsin/EDTA solution (Cascade Biologics), neutralized by defined 0.0125% trypsin inhibitor (Cascade Biologics), centrifuged, and resuspended at a seeding density of 1.0 × 10 4 cells/cm 2 . Passage three cells were used for assaying colony forming efficiency and making the survival curve for ionizing radiation.
Fabrication of EVPOME model
The development of human EVPOME was established according to the protocol described previously . Briefly, passage three cells were used for fabrication of human EVPOME. Circular pieces of AlloDerm ® (LifeCell, Branchburg, NJ, USA), 1.0 cm in diameter, were rehydrated by sterile PBS and the surface was coated with 5 μg/cm 2 collagen type IV (BD Biosciences, Flanklin Lakes, NJ, USA) at 4 °C in a refrigerator overnight. Oral keratinocytes were seeded onto AlloDerm ® at a density of 1.2–1.5 × 10 5 /cm 2 and cultured at a high calcium concentration of 1.2 mM under submerged condition for 4 days. The equivalent was raised to an air–liquid interface culture plate (Organogenesis Inc., Canton, MA, USA) with high calcium concentration and cultured for seven additional days, for a total culture time of 11 days.
Ionizing irradiation of EVPOMEs
X-ray irradiation was performed using Therapax DXT300 X-ray radiation source (Pantak, Easthaven, CT, USA). The focus-culture plate distance was 50 cm and the dose rate was approximately 3 Gy/min. Control plates were sham-irradiated by placing them on the radiation table without irradiating. All EVPOMEs and monolayer cultures of oral mucosa keratinocyte were kept out of the incubator during irradiation (for approximately 15 min). The dose applied was 0, 1, 3 and 8 Gy in the form of X photons of 300 kV. These doses were determined by a pilot study. Irradiated and control EVPOMEs and monolayer cultures of oral mucosa keratinocyte were immediately returned to the incubator at 37 °C in a humidified environment and cultured for an additional 24 h.
Spent culture media and EVPOMEs were taken for samples of each assay. Spent cultured media was taken and stored in a freezer at −20 °C. All spent culture medium samples were processed through an ELISA at the same time to assess the presence of the pro-inflammatory cytokines, IL-1α and IL-8. EVPOME was cut in half and one piece was fixed with 10% formalin for histopathological examination and immunohistochemistry, while the other was used immediately for cell viability assay.
Colony forming efficiency (CFE) assay
CFE for cell proliferation was determined by plating irradiated cells in a 60 mm culture dish at a density of 500 cells/cm 2 using monolayer cultures of oral mucosa keratinocyte. Cells were irradiated with 0, 0.5, 1, 3, 5, and 8 Gy and cultured for 10–12 days. Colonies larger than 16 cells were counted under ×100 magnification with a phase contrast microscope, and each dose of CFE fraction was calculated using Eq. (1) :
CFE = the plating efficiency of the irradiated culture the plating efficiency of the control culture