the pathogenesis of middle ear cholesteatoma has been debated for a century , but there are four basic theories : invagination , basal cell hyperplasia , epithelial ingrowth and squamous metaplasia . during last century , etiopathogenicity of middle ear cholesteatoma raised many controversies and still a unanimous concept does n't exist . from a histological point of view this pathological tissue
the interaction between cellular components is essential for the major histopathological features of cholesteatoma - hyperproliferation and bone destruction . in 1964 , the american anatomist gray referred to middle ear cholesteatoma as " skin in the wrong place " ( 1 ) .
this simplified misdefinition stuck into literature and has been cited profusely in many scientific papers . the metaplasia theory ( 2 ) and existence of so - called " congenital " cholesteatoma put into question the " skin origin " of middle ear cholesteatoma and gave etiopathogenetical evidences against the simplified definition " skin in the wrong place " .
the list of histological differences includes the absence of cutaneous adnexa and specialized dermal structures .
neither hypodermal structures , nor blood vessels , or nerve endings have been ever described in cholesteatoma .
the only similarity that is left between them is the presence of multistratified squamous epithelium , but in cholesteatoma this epithelium is with hyperproliferative properties .
experimental models are critical for studying pathogenesis of middle ear cholesteatoma . in vivo models were tried to establish by chemical injection into the middle ear ( 3 ) , ligation of external auditory canal ( 4 ) and transplantation of skin ( 5 ) .
keratinocytes of middle ear cholesteatoma were cultured by tissue culture ( 6 ) , passaged subculture ( 7 ) and air - liquid interface system ( 8) . however , stable experimental models of middle ear cholesteatoma are not established until now .
our study has aimed to create an " adequate " in vitro model , based on the cell - to - cell interaction between keratinocytes and fibroblasts and to assess the grade of homogeneity between cholesteatoma and skin keratinocytes by focusing on the cytokeratin profile .
enzymatic disaggregation protocol for simultaneous isolation of primary keratinocytes and fibroblasts from cholesteatoma tissue has been developed .
the biopsy samples were harvested from patients undergoing surgical treatment in the department of otolaryngology , ajou university hospital , korea . an informed consent form approved by the institutional review board of ajou university hospital
cholesteatoma keratinocytes were cultured in defined keratinocytes serum free medium ( gibco , brl , gaithersburg , md , usa ) supplied with growth supplement and 5 mg / ml gentamicin ( gibco , brl ) according to the manufacture instructions .
cholesteatoma primary fibroblasts were successfully cultured in dulbeco modified eagle medium ( gibco , brl ) supplemented with 10% fetal bovine serum ( fbs , clonetics , walkersville , md , usa ) and 1% penicillin - streptomycin mixture ( gibco , brl ) .
primary keratinocytes ( passage 1 ) and fibroblasts ( between passages 1 - 5 ) were used for establishment of the co - cultured system .
hacat cell line was kindly provided from the department of dermatology in ajou university hospital . feeder layer from cholesteatoma fibroblasts
louis , mo , usa ) in concentration 0.25 g / ml . fibroblasts were incubated overnight at 37 , 5% co2 in humidified atmosphere . on the next day
the feeder layer was subsequently trypsinized from the substrate and transferred on polyester membrane in transwell ( costar , cambridge , ma , usa ) system to develop a subconfluent feeder layer .
cholesteatoma keratinocytes and hacat cells were seeded at density 210 cells / ml over the feeder layer , co - cultured for 3 - 5 days and air - exposed for a period of 14 days .
transwell membranes were fixed in 4% formaldehyde overnight at 4 , dehydrated in graded alcohols , cleared in histoclear ( national diagnostics , atlanta , georgia ) and embedded in paraffin .
six m - thick sections were prepared and subjected to routine hematoxylin - eosin staining .
polyacrylamide gel electrophoresis has been used for protein separation and staining with coomassie blue was performed to compare the protein profile between cholesteatoma keratinocytes and hacat cells .
blocking of nonspecific staining was accomplished with membrane incubation with 5% non - fat skim milk for 1 hr at room temperature ( rt ) .
membranes were then incubated with primary mouse monoclonal anti - cytokeratin antibody clone k 8.12 ( sigma ) which is immunospecific for cytokeratin 13 ( ck 13 ) and cytokeratin 16 ( ck 16 ) , with dilution factor 1:100 , for 1 hr at rt .
after washing membranes were incubated with goat anti - mouse horseradish peroxidase conjugate ( zymed , san francisco , ca , usa ) .
the positive reaction was visualized with ecl western blotting detection system ( amersham , bucks , uk ) .
the localization of cytokeratin 13 and 16 in middle ear cholesteatoma tissue was accomplished by ihc study as described before ( 9 ) .
five middle ear cholesteatoma samples were compared with the corresponding retroauricular skin samples obtained during surgery .
six m thick formalin - fixed paraffin - embedded tissue sections were processed with abc - method .
primary mouse monoclonal anticytokeratin antibody ( sigma ) at concentration 1:20 was incubated overnight at rt .
vectorlab elite abc kit ( vector laboratories , burlingame , ca , usa ) was used for detection of positive reaction using diaminobenzidine substrate .
enzymatic disaggregation protocol for simultaneous isolation of primary keratinocytes and fibroblasts from cholesteatoma tissue has been developed .
the biopsy samples were harvested from patients undergoing surgical treatment in the department of otolaryngology , ajou university hospital , korea . an informed consent form approved by the institutional review board of ajou university hospital
cholesteatoma keratinocytes were cultured in defined keratinocytes serum free medium ( gibco , brl , gaithersburg , md , usa ) supplied with growth supplement and 5 mg / ml gentamicin ( gibco , brl ) according to the manufacture instructions .
cholesteatoma primary fibroblasts were successfully cultured in dulbeco modified eagle medium ( gibco , brl ) supplemented with 10% fetal bovine serum ( fbs , clonetics , walkersville , md , usa ) and 1% penicillin - streptomycin mixture ( gibco , brl ) .
primary keratinocytes ( passage 1 ) and fibroblasts ( between passages 1 - 5 ) were used for establishment of the co - cultured system .
hacat cell line was kindly provided from the department of dermatology in ajou university hospital . feeder layer from cholesteatoma fibroblasts
louis , mo , usa ) in concentration 0.25 g / ml . fibroblasts were incubated overnight at 37 , 5% co2 in humidified atmosphere . on the next day
the feeder layer was subsequently trypsinized from the substrate and transferred on polyester membrane in transwell ( costar , cambridge , ma , usa ) system to develop a subconfluent feeder layer .
cholesteatoma keratinocytes and hacat cells were seeded at density 210 cells / ml over the feeder layer , co - cultured for 3 - 5 days and air - exposed for a period of 14 days .
transwell membranes were fixed in 4% formaldehyde overnight at 4 , dehydrated in graded alcohols , cleared in histoclear ( national diagnostics , atlanta , georgia ) and embedded in paraffin .
six m - thick sections were prepared and subjected to routine hematoxylin - eosin staining .
polyacrylamide gel electrophoresis has been used for protein separation and staining with coomassie blue was performed to compare the protein profile between cholesteatoma keratinocytes and hacat cells .
blocking of nonspecific staining was accomplished with membrane incubation with 5% non - fat skim milk for 1 hr at room temperature ( rt ) .
membranes were then incubated with primary mouse monoclonal anti - cytokeratin antibody clone k 8.12 ( sigma ) which is immunospecific for cytokeratin 13 ( ck 13 ) and cytokeratin 16 ( ck 16 ) , with dilution factor 1:100 , for 1 hr at rt .
after washing membranes were incubated with goat anti - mouse horseradish peroxidase conjugate ( zymed , san francisco , ca , usa ) .
the positive reaction was visualized with ecl western blotting detection system ( amersham , bucks , uk ) .
the localization of cytokeratin 13 and 16 in middle ear cholesteatoma tissue was accomplished by ihc study as described before ( 9 ) .
five middle ear cholesteatoma samples were compared with the corresponding retroauricular skin samples obtained during surgery .
six m thick formalin - fixed paraffin - embedded tissue sections were processed with abc - method .
primary mouse monoclonal anticytokeratin antibody ( sigma ) at concentration 1:20 was incubated overnight at rt .
vectorlab elite abc kit ( vector laboratories , burlingame , ca , usa ) was used for detection of positive reaction using diaminobenzidine substrate .
the low selectivity and potential damages of cells in case of prolonged incubation were major disadvantages of the procedure .
as an inflammatory tissue , cholesteatoma posed some difficulties in isolation of primary keratinocytes , because of the attendant inflammation and possible contamination , which requires an adequate decontamination .
the first primary keratinocytes appeared usually 7 - 10 days after trypsinization and generally within the next 10 days keratinocytes reached a confluence .
the individual variations in proliferative activity of cholesteatoma keratinocytes were related to cholesteatoma localization and sex of the patient .
for instance , attic cholesteatoma frequently resulted in isolation of primary keratinocytes with bigger proliferative ability .
another interesting correlation was the fact that cholesteatoma samples obtained from young female patients were characterized by higher proliferative activity in vitro .
primary cholesteatoma keratinocytes were uniform in shape and arranged in a typical monolayer pattern ( fig .
in passage 2 an abrupt deceleration in proliferation was established , accompanied by morphological alterations such as enlargement and flattening of the cells , as well as vacuolization of the cytoplasm . in passage 3
primary cholesteatoma fibroblasts were steadily maintained without significant changes of doubling time until passage 10 .
bigger stratification and proliferation have been observed in the proposed 3d in vitro system when primary cholesteatoma keratinocytes were co - cultured in the presence of fibroblasts ( fig .
3b ) in comparison with the control group without fibroblasts ( fig . 3a ) .
4a ) . disrupted differentiation in the suprabasal layers was revealed when cholesteatoma keratinocytes were air - exposed in the presence of fibroblasts mimicking the parakeratosis state , which is typical for this pathological tissue in vivo ( fig .
coomassie blue staining of the protein extracts from primary cholesteatoma keratinocytes and hacat cells revealed significantly different protein profiles ( fig .
stronger expression of ck 16 ( 48 kda ) and ck 13 ( 51 kda ) was established in cholesteatoma keratinocytes in comparison with hacat cells ( fig .
5b ) . using mouse monoclonal antibody clone k 8.12 , tissue localization of ck 13 and ck 16
the intensity of the ck 13 , 16-positive staining varied in different areas of the same cholesteatoma sample .
the positive control revealed specific expression of ck 13 and 16 confined to the islands of neoplastic squamous cells in laryngeal carcinoma specimens .
the low selectivity and potential damages of cells in case of prolonged incubation were major disadvantages of the procedure .
as an inflammatory tissue , cholesteatoma posed some difficulties in isolation of primary keratinocytes , because of the attendant inflammation and possible contamination , which requires an adequate decontamination .
the first primary keratinocytes appeared usually 7 - 10 days after trypsinization and generally within the next 10 days keratinocytes reached a confluence .
the individual variations in proliferative activity of cholesteatoma keratinocytes were related to cholesteatoma localization and sex of the patient .
for instance , attic cholesteatoma frequently resulted in isolation of primary keratinocytes with bigger proliferative ability .
another interesting correlation was the fact that cholesteatoma samples obtained from young female patients were characterized by higher proliferative activity in vitro .
primary cholesteatoma keratinocytes were uniform in shape and arranged in a typical monolayer pattern ( fig .
in passage 2 an abrupt deceleration in proliferation was established , accompanied by morphological alterations such as enlargement and flattening of the cells , as well as vacuolization of the cytoplasm . in passage 3
primary cholesteatoma fibroblasts were steadily maintained without significant changes of doubling time until passage 10 .
bigger stratification and proliferation have been observed in the proposed 3d in vitro system when primary cholesteatoma keratinocytes were co - cultured in the presence of fibroblasts ( fig .
3b ) in comparison with the control group without fibroblasts ( fig . 3a ) .
4a ) . disrupted differentiation in the suprabasal layers was revealed when cholesteatoma keratinocytes were air - exposed in the presence of fibroblasts mimicking the parakeratosis state , which is typical for this pathological tissue in vivo ( fig .
coomassie blue staining of the protein extracts from primary cholesteatoma keratinocytes and hacat cells revealed significantly different protein profiles ( fig .
ck 13 ( 51 kda ) was established in cholesteatoma keratinocytes in comparison with hacat cells ( fig .
using mouse monoclonal antibody clone k 8.12 , tissue localization of ck 13 and ck 16 was verified both in the basal and suprabasal layers of cholesteatoma ( fig .
the intensity of the ck 13 , 16-positive staining varied in different areas of the same cholesteatoma sample .
the positive control revealed specific expression of ck 13 and 16 confined to the islands of neoplastic squamous cells in laryngeal carcinoma specimens .
the putative interactions between major cellular components of cholesteatoma tissue have been extensively studied in the literatures .
we demonstrate a summarized schematic model of possible intercellular " cross - talking " between epithelial - mesenchymal components of cholesteatoma in fig .
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 differentiation of keratinocytes ( 11 ) .
keratinocytes - derived parathyroid hormone - related protein ( pthrp ) induces expression of kgf .
kgf is then released by perimatrix fibroblasts and accelerates the proliferative response in matrix keratinocytes .
a possible connection between pthrp and bone destructive lesions have also been reported ( 12 ) . transforming growth factor alpha ( tgf- )
is constitutively expressed in keratinocytes and regulates keratinocyte proliferation and differentiation in an autocrine manner ( 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 alternative paracrine regulatory mechanism ( 14 ) .
transforming growth factor - beta ( tgf- ) is expressed in hyperproliferative epithelium and granulation tissue ( 15 ) and influences the epithelial 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 proliferative capacity of primary cholesteatoma keratinocytes in vitro is limited and expires within 2 passages . to improve their plating efficiency and survival rate at low density
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 normal skin keratinocytes .
hacat cells are spontaneously immortalized human adult skin keratinocytes raised in reduced calcium 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 stratification , when hacat cells were co - cultured with fibroblasts , in comparison with the control group without fibroblasts .
histopathological diagnosis of cholesteatoma includes : unrestrained proliferation , uncoordinated differentiation , migration , and invasion ( 18 ) .
consistent with the above - mentioned observation , the proposed 3d - model disclosed disrupted differentiation and presence of nucleated cells in the upper layers of the cholesteatoma ali with fibroblasts .
squamous metaplasia of middle ear epithelium is considered to be an adaptive tissue response towards persistent pathological stimuli such as chronic inflammation and possible etiopathogenetical mechanism for middle ear cholesteatoma .
an experiment has been reported on the confirmation of a possible transformation of differentiated middle ear epithelium towards functionally inferior multilayered squamous epithelium ( 19 ) .
epithelial cells are characterized by the presence of a specific set of intermediate protein filaments named cytokeratins , specific for different epithelial types .
one of our primary objectives was to assess the grade of homogeneity between hacat cells and cholesteatoma keratinocyte .
ck 16 was suggested in the literature as a proliferation marker , while ck 13 is considered as a marker of differentiation ( 20 ) .
immunohistochemical investigation in our study disclosed non - uniform patterns of expression of ck 13 and ck 16 in middle ear cholesteatoma tissue .
variation in intensity of the positively stained areas between different cholesteatoma samples as well as between different areas in the same sample was probably related to the different proliferative capacity of cholesteatoma .
the expression of nonepidermal cytokeratins in middle ear cholesteatoma tissue gives arguments against the probable skin origin of mech keratinocytes .
protein separation and coomassie blue staining revealed significantly different protein profile between cholesteatoma and hacat cells .
precised western - blotting analysis demonstrated comparatively stronger expression of ck 13 and 16 in cholesteatoma keratinocytes with regard to hacat cell line .
the different protein and cytokeratin profiles established between cholesteatoma keratinocytes and hacat cells favors metaplasia as a possible cause for development of mech .
the proposed in vitro model underlines the importance of fibroblasts as a major component of 3d - in vitro system .
our results emphasize the integrity of cholesteatoma as a separated pathological tissue , composed of two closely related functional and structural parts : the matrix and the perimatrix .
 objectivesexperimental models are of importance to study the pathogenesis of middle ear cholesteatoma , however , they were not established until now . we aimed to develop in vitro model of middle ear cholesteatoma using primary keratinocytes and fibroblasts isolated from cholesteatoma tissue . 
 hacat cell line was used as a " skin equivalent " and to compare the grade of homogeneity between cholesteatoma keratinocytes and hacat cells.methodsprimary keratinocytes were isolated from cholesteatoma tissue , co - cultured with preliminary prepared feeder layer from cholesteatoma fibroblasts and subsequently air - exposed . 
 the protein profile of cholesteatoma keratinocytes and hacat cells was evaluated by means of immunoblot using monoclonal antibody against cytokeratin ( ck ) 13 and 16 . 
 tissue localization of ck 13 and 16 was accomplished with immunohistochemistry.resultsdifferent protein profile and stronger expression of ck 13 and 16 were demonstrated in cholesteatoma keratinocytes in comparison with hacat cells . 
 bigger stratification was observed in the 3d - in vitro systems when both cholesteatoma keratinocytes and hacat cells were respectively co - cultured with fibroblasts in comparison with the corresponding control groups without fibroblasts.conclusion3d-model demonstrates the significance of intercellular interaction between components of cholesteatoma tissue . 
INTRODUCTION
MATERIALS AND METHODS
Cell culture
Morphological study
Protein profile
Cytokeratin profile
Immunohistochemistry (IHC)
RESULTS
Cell culture
Air-liquid interface (ALI)
Protein and cytokeratin profile
Immunohistochemistry
DISCUSSION
CONCLUSIONS