90 Clinical and Experimental Otorhinolaryngology Vol. 1, No. 2: 86-91, June 2008 The intensity of the CK 13, 16-positive staining varied in differ- ent areas of the same cholesteatoma sample. Specific positive reaction was not detected in retroauricular skin samples used as a control. The positive control revealed specific expression of CK 13 and 16 confined to the islands of neoplastic squamous cells in laryngeal carcinoma specimens. DISCUSSION The putative interactions between major cellular components of cholesteatoma tissue have been extensively studied in the lit- eratures. We demonstrate a summarized schematic model of possible intercellular “cross-talking” between epithelial-mesenchy- mal components of cholesteatoma in Fig. 7. Both paracrine and autocrine regulatory mechanisms have been proposed for main- tenance of tissue homeostasis. Pro-inflammatory cytokines such as interleukin-1 and interleukin-1 ‚ (IL-1 , IL-1 ) released from matrix keratinocytes stimulate keratinocytes growth factor (KGF) expression, which in turn induced proliferation of the cholesteatoma keratinocytes (10). Granulocyte-macrophage colony stimulating factor (GM-CSF) is another fibroblasts- derived growth factor induced by IL-1, with acknowledged stimulating effects both on proliferation and terminal differentia- tion of keratinocytes (11). Keratinocytes-derived parathyroid hor- mone-related protein (PTHrP) induces expression of KGF. KGF is then released by perimatrix fibroblasts and accelerates the pro- liferative response in matrix keratinocytes. A possible connec- tion between PTHrP and bone destructive lesions have also been reported (12). Transforming growth factor alpha (TGF- ) is constitutively expressed in keratinocytes and regulates ker- atinocyte proliferation and differentiation in an autocrine man- IL-1 PTHrP TGF , KGF, EGF GM-CSF Fig. 7. Summarized schematic model of putative autocrine and paracrine regulatory mechanisms, responsible for maintaining of middle ear cho- lesteatoma tissue homeostasis. IL: interleukin; PTHrP: parathyroid hor- mone-related protein; TGF: transforming growth factor; KGF: kerati- nocytes growth factor; EGF: epidermal growth factor; GM-CSF: gran- ulocyte-macrophage colony stimulating factor. ner (13). On the other hand, a possible synergism between TGF- and epidermal growth factor (EGF) was proposed with possible effect on keratinocytes proliferation through epidermal growth factor receptor (EGF-R), indicating existence of an alter- native paracrine regulatory mechanism (14). Transforming growth factor-beta (TGF- ) is expressed in hyperproliferative epithelium and granulation tissue (15) and influences the epithe- lial morphogenesis both in an autocrine and paracrine fashion. The above statements about cellular interactions were taken into consideration for creating an adequate in vitro middle ear cholesteatoma model. As it was mentioned before, the prolifer- ative capacity of primary cholesteatoma keratinocytes in vitro is limited and expires within 2 passages. To improve their plat- ing efficiency and survival rate at low density we create a growth- arrested feeder layer from primary middle ear cholesteatoma fibroblasts. 3T3-mouse embryo fibroblasts that used routinely in cell culture practice display different morphological patterns (cobble-stone morphology), which differs from in vivo state and complicates result interpretation. Feeder layer secretes nutrients, growth factors and matrix constituents. As a positive control in our study we choose HaCaT cell line in order to avoid donor-to- donor and site-to-site variations in proliferative capacity of nor- mal skin keratinocytes. HaCaT cells are spontaneously immor- talized human adult skin keratinocytes raised in reduced calci- um level and elevated temperature conditions (16). HaCaT found an extensive application in tissue engineering for development of artificial skin equivalents (17). The proposed organotypic in vitro model revealed stronger proliferation and bigger stratifi- cation, when HaCaT cells were co-cultured with fibroblasts, in comparison with the control group without fibroblasts. Similar patterns have been established with primary cholesteatoma ker- atinocytes. Histopathological diagnosis of cholesteatoma includes: unrestrained proliferation, uncoordinated differentiation, migra- tion, and invasion (18). Consistent with the above-mentioned observation, the proposed 3D-model disclosed disrupted differ- entiation and presence of nucleated cells in the upper layers of the cholesteatoma ALI with fibroblasts. The following data cor- responds to the parakeratotic properties in cholesteatoma tissue in vivo. Squamous metaplasia of middle ear epithelium is considered to be an adaptive tissue response towards persistent pathologi- cal stimuli such as chronic inflammation and possible etiopatho- genetical mechanism for middle ear cholesteatoma. An experi- ment has been reported on the confirmation of a possible trans- formation of differentiated middle ear epithelium towards func- tionally inferior multilayered squamous epithelium (19). Epithelial cells are characterized by the presence of a specific set of inter- mediate protein filaments named cytokeratins, specific for dif- ferent epithelial types. One of our primary objectives was to assess the grade of homogeneity between HaCaT cells and cho- lesteatoma keratinocyte. CK 16 was suggested in the literature as a proliferation marker, while CK 13 is considered as a mark-