ABSTRACT
The aim of this study was to evaluate the suitability of tissue-engineered mucosa (TEM) as a model for studying the acute effects of ionizing radiation (IR) on the oral mucosa. TEM and native non-keratinizing oral mucosa (NNOM) were exposed to a single dose of 16.5Gy and harvested at 1, 6, 24, 48, and 72h post-irradiation. DNA damage induced by IR was determined using p53 binding protein 1 (53BP1), and DNA repair was determined using Rad51. Various components of the epithelial layer, basement membrane, and underlying connective tissue were analyzed using immunohistochemistry. The expression of cytokines interleukin-1ß (IL-1ß) and transforming growth factor beta 1 (TGF-ß1) was analyzed using an enzyme-linked immunosorbent assay. The expression of DNA damage protein 53BP1 and repair protein Rad51 were increased post-irradiation. The expression of keratin 19, vimentin, collage type IV, desmoglein 3, and integrins α6 and ß4 was altered post-irradiation. Proliferation significantly decreased at 24, 48, and 72h post-irradiation in both NNOM and TEM. IR increased the secretion of IL-1ß, whereas TGF-ß1 secretion was not altered. All observed IR-induced alterations in TEM were also observed in NNOM. Based on the similar response of TEM and NNOM to IR we consider our TEM construct a suitable model to quantify the acute biological effects of IR.
Subject(s)
Mouth Mucosa/radiation effects , Tissue Engineering , Basement Membrane/radiation effects , Cell Adhesion/radiation effects , Cell Culture Techniques , Cell Differentiation/radiation effects , Cell Proliferation/radiation effects , Collagen Type IV/analysis , Collagen Type IV/radiation effects , Connective Tissue/radiation effects , DNA Damage/radiation effects , DNA Repair/radiation effects , Desmoglein 3/analysis , Desmoglein 3/radiation effects , Epithelium/radiation effects , Female , Fibroblasts/radiation effects , Gamma Rays , Humans , Integrin alpha6/analysis , Integrin alpha6/radiation effects , Integrin beta4/analysis , Integrin beta4/radiation effects , Interleukin-1beta/analysis , Interleukin-1beta/radiation effects , Intracellular Signaling Peptides and Proteins/analysis , Intracellular Signaling Peptides and Proteins/radiation effects , Keratin-19/analysis , Keratin-19/radiation effects , Keratinocytes/radiation effects , Male , Middle Aged , Mouth Mucosa/cytology , Rad51 Recombinase/analysis , Rad51 Recombinase/radiation effects , Radiation Dosage , Transforming Growth Factor beta1/analysis , Transforming Growth Factor beta1/radiation effects , Tumor Suppressor p53-Binding Protein 1 , Vimentin/analysis , Vimentin/radiation effectsABSTRACT
In this study we have employed atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM) techniques to study the effect of the interaction between human keratinocytes (HaCaT) and electromagnetic fields at low frequency. HaCaT cells were exposed to a sinusoidal magnetic field at a density of 50 Hz, 1 mT. AFM analysis revealed modification in shape and morphology in exposed cells with an increase in the areas of adhesion between cells. This latter finding was confirmed by SNOM indirect immunofluorescence analysis performed with a fluorescent antibody against the adhesion marker beta4 integrin, which revealed an increase of beta4 integrin segregation in the cell membrane of 50-Hz exposed cells, suggesting that a higher percentage of these cells shows a modified pattern of this adhesion marker.
