Epithelial–mesenchymal interactions are thought to play an important role in the pathogenesis of odontogenic lesions. Keratocystic odontogenic tumour (KCOT) is a benign cystic neoplasm with a characteristic parakeratinized epithelial lining, which differs histologically and behaviourally from the so-called orthokeratinized odontogenic cyst (OOC). The purpose of this study was to investigate the differences in collagen fibres within the fibrous tissue walls of KCOT and OOC. Formalin-fixed paraffin-embedded tissue samples from 15 cases of KCOT and 15 cases of OOC were collected. Paraffin sections were stained with picrosirius red and observed under a standard light microscope using optical polarization. Unicystic ameloblastoma (UA, 15 cases) and subcutaneous epidermoid cysts (EC, 15 cases) were included in the study for comparative purposes. Significant difference was detected between the polarization colours in the fibrous tissue walls of KCOT and OOC ( P < 0.05), whilst no significant differences were found between KCOT and UA and between OOC and EC ( P > 0.05). The stromal collagen fibres of KCOT were different from those of OOC, but similar to those of UA, which suggests that the stroma of KOCT may play an important role in determining the neoplastic behaviour of the lesion through epithelial–mesenchymal interaction.
Keratocystic odontogenic tumour (KCOT) is a benign, intraosseous tumour of odontogenic origin, with a characteristic lining of parakeratinized stratified squamous epithelium and locally aggressive behaviour. Some are associated with nevoid basal cell carcinoma syndrome. The traditional designation is odontogenic keratocyst (OKC), which stresses the cystic nature of the lesion . Orthokeratinized odontogenic cyst (OOC) is a rare intraosseous cyst characterized by an orthokeratinized epithelium lining, and which shows little clinical aggressiveness . According to the new World Health Organization classification, cystic jaw lesions lined by orthokeratinized epithelium do not form part of the spectrum of KCOT and should thus be distinguished from the latter. Several studies have indicated that the lining epithelium of OOC lacked the typical features of KCOT and appeared to show lower proliferative activity . Reciprocal interaction between dental epithelium and mesenchyme is thought to be crucial for normal odontogenesis. Thus the mesenchymal influence of the fibrous capsules may play an important role in the maintenance of epithelial expression of KCOT and OOC .
Picrosirius red staining in combination with polarization microscopy has been used to study individual collagen fibres and to determine their content in the specific tissue. Collagen molecules, being rich in basic amino acids and disposed in a parallel orientation, can strongly react with acidic dyes. Sirius red is an elongated dye molecule which reacts with collagen and promotes an enhancement of its normal birefringence due to the fact that many dye molecules are aligned parallel with the long axis of each collagen molecule. The enhancement of birefringence promoted by the picrosirius polarization method is specific for collagenous structures composed of aggregates of orientated molecules .
Both fibre thickness and packing of collagen can cause differences in polarization colours . Examination of collagen fibres by this method can serve as a procedure to differentiate precollagens, intermediate and pathological collagen fibres, which are not tightly packed, from normal packed fibres. This has been shown to be of value in human tooth germ papillae , skin lesions , odontogenic tumours and odontogenic cysts . Using picrosirius red staining, H irshberg et al. have shown that the polarization colour pattern of collagen fibres in the connective tissue stroma of KCOT differs from dentigerous and radicular cysts.
The aim of this study was to compare the collagen fibres within the fibrous tissue walls of KCOT and OOC using picrosirius red staining and polarizing microscopy. Unicystic ameloblastomas (UAs) and similar soft tissue epidermoid cysts (EC) were studied for comparison.
Materials and methods
583 cases coded as KCOT were reviewed from the files of the authors’ department during the period 1985–2008. After reviewing the patient details, clinical information and histology, 61 OOC cases were identified based on previously established criteria . For inclusion in the OCC group, all or a predominant portion of the lining epithelium had to exhibit orthokeratinization, in which the basal cells showed no tendency to palisade. A clinical and pathological study of these 61 cases has been previously reported . For the present study, 15 more recent cases were selected from this OOC group. 15 cases of KCOT showing typical parakeratinized lining epithelium, 15 cases of UA (simple cyst type without intraluminal or mural tumour growth) and 15 cases of soft tissue EC were also included in the study for comparative purposes. Histologically, the lining epithelium of EC is almost identical to that of OOC although EC occurs in the soft tissues. It would be interesting to compare their fibrous capsules. All samples were formalin-fixed and paraffin wax-embedded for routine histological examination.
Picrosirius red staining
For visualization of the collagen fibres, 4-μm thick sections were stained with picrosirius red. In brief, after deparaffinization in xylene and ethanol, sections were hydrated in distilled water, followed by incubation in sirius red (0.1% in saturated picric acid, Electron Microscopy Sciences, USA) for 1 h at room temperature, rinsed with distilled water, and counter stained with haematoxylin. Sections were examined by polarizing microscopy (BX51, Olympus, Japan). Collagen fibres were illustrated as orange-red and/or green colour.
Examination of the polarized colours of stained collagen fibres
To correct potential variability in the staining intensity of sections from different staining batches, all image-acquisition parameters were fixed during the process of image-capture, and the intensity of acquisition illumination was calibrated by adjusting only the microscope condenser aperture. The evaluation process was as follows. Mature and immature collagen fibres were differentiated by the polarization colours. Against a black background, thick yellowish-red fibres were mainly mature collagen (MC), whilst fine netlike greenish-yellow fibres were mainly immature collagen (IC) . The images were evaluated by an image analyzer (Image Pro Plus 6.0, Media Cybernetics Inc., Silver Spring, MD, USA), which can automatically calculate the area of the defined regions (yellow-red or green) in each section. Results were expressed as area of mature (SM) and immature (SI) collagen fibres. The ratios of SM/SI were calculated to represent the content of mature and immature collagen fibres. In the preliminary experiment, the authors found that reproducible values, not affected by further counts, were achieved by observing eight non-inflammation and non-overlapping regions of capsule wall. Therefore, in each section, eight regions at 200× magnification were analysed.
For the image obtained by polarized microscopy, segmentation was done and the desired pseudo colour image was obtained by substitution with pure black colour. For MC, the orange-red part was obtained by segmentation and the rest of the image was substituted with pure black, which had a grey level of zero. Thus any pixel with a grey-scale level greater than zero represented MC. The image was converted to an eight-bit grey image, and the result of the conversion was an image consisting of grey collagen fibres on a black background. A histogram of the brightness of each pixel in the image was plotted. Thus, collagen content could be expressed as the mean fraction of pixels with a grey-scale level greater than zero. For the IC, a similar procedure was followed. After segmentation, substitution, and subtraction, the green part of the image was converted to an eight-bit grey level for quantification. The ratio of MC to IC was calculated . The authors also observed eight non-inflammation and non-overlapping regions of each section (at 200× magnification) for analysis.
To eliminate subjective bias, images were evaluated independently by two observers. Data were expressed as mean ± standard error of the mean. Statistical differences between groups were analysed using SPSS 13.0 software (SPSS Inc, Chicago, Illinois, USA). One-way Analysis of Variance (AVONA) was also carried out. Differences were considered to be significant when P < 0.05.
The tissue sections were observed with polarized microscopy after picrosirius red staining. In sections of KCOT and UA, a greenish-yellow polarization colour predominated in the subepithelial areas of the fibrous capsule. In OOC and EC, a yellowish-red polarization colour predominated in the subepithelial zone ( Fig. 1 a–h).