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The objective of this study was to evaluate the expression of matrix metalloproteinase 9 (MMP-9) and transforming growth factor beta (TGF-ß1) in periapical lesion samples correlated with the intensity of the inflammatory infiltrate and thickness of the epithelial lining. Forty-five cases of periapical lesions (23 periapical granulomas and 22 radicular cysts) were subjected to morphological and immunohistochemical analyses using anti-MMP-9 and anti-TGF-ß1 antibodies. The data were analyzed using the following tests: non-parametric Mann-Whitney, chi-square, Fisher's exact test and Spearman's correlation test (P<0.05). Analysis of inflammatory infiltrate revealed that 78% of periapical granulomas presented infiltrate grade III, in contrast with 32% of radicular cysts (P<0.001). Morphological evaluation of the epithelial thickness in radicular cysts revealed the presence of atrophic epithelium in 86% of the cysts. The immunostaining of MMP-9 was score 2 in 67% of the granulomas and 77% of the cysts. Both lesions were predominantly score 1 for TGF-ß1. Significant differences were confirmed between the expression scores of TGF-ß1 and MMP-9 in periapical granulomas (p = 0.004) and in radicular cysts (p < 0.001). Expression of TGF-ß1 was different for periapical granulomas and radicular cysts. This immunoregulatory cytokine seems more representative in asymptomatic lesions. The extracellular matrix remodeling process dependent on MMP-9 seems to be similar for both periapical granulomas and radicular cysts. TGF-ß1 and MMP-9 may play an important role in the maintenance of periapical lesions.

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Abstract The objective of this study was to evaluate the expression of matrix metalloproteinase 9 (MMP-9) and transforming growth factor beta (TGF-β1) in periapical lesion samples correlated with the intensity of the inflammatory infiltrate and thickness of the epithelial lining. Forty-five cases of periapical lesions (23 periapical granulomas and 22 radicular cysts) were subjected to morphological and immunohistochemical analyses using anti-MMP-9 and anti-TGF-β1 antibodies. The data were analyzed using the following tests: non-parametric Mann-Whitney, chi-square, Fisher's exact test and Spearman's correlation test (P<0.05). Analysis of inflammatory infiltrate revealed that 78% of periapical granulomas presented infiltrate grade III, in contrast with 32% of radicular cysts (P<0.001). Morphological evaluation of the epithelial thickness in radicular cysts revealed the presence of atrophic epithelium in 86% of the cysts. The immunostaining of MMP-9 was score 2 in 67% of the granulomas and 77% of the cysts. Both lesions were predominantly score 1 for TGF-β1. Significant differences were confirmed between the expression scores of TGF-β1 and MMP-9 in periapical granulomas (p = 0.004) and in radicular cysts (p < 0.001). Expression of TGF-β1 was different for periapical granulomas and radicular cysts. This immunoregulatory cytokine seems more representative in asymptomatic lesions. The extracellular matrix remodeling process dependent on MMP-9 seems to be similar for both periapical granulomas and radicular cysts. TGF-β1 and MMP-9 may play an important role in the maintenance of periapical lesions.

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PURPOSE: To determine the biological behaviour of common odontogenic cystic lesions by analysing and comparing bcl-2 expression amongst them. MATERIALS AND METHODS: Our study covered 90 formalin fixed paraffin embedded tissue samples: 26 primary cases each of radicular cysts (RC), dentigerous cysts (DC) and odontogenic keratocysts (OKC) and 12 of recurrent OKCs. Bcl-2 expression was analysed immunohistochemically and data analysis was accomplished using SPSS version 17.0. Means were taken for age while for gender and site of the lesions frequencies and percentages were determined. The Chi-square test was applied to evaluate any statistically significant difference of bcl-2 expression in these lesions and p value of ≤0.05 was taken as significant. RESULTS: All the recurrent OKCs showed a strong positivity for bcl-2 that was absent in all of its primary cases (p value<0.05). Although variation in expression of bcl-2 was not found to be statistically significant between RC and DC, however, it became significant when all primary cases of these common odontogenic lesions were compared. CONCLUSIONS: Recurrent OKC showed comparatively a more aggressive behaviour than their primary counterparts and also from RC and DC. Bcl-2 proved to be a valuable adjunct in determining aggressive biological behaviour of odontogenic lesions.

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OBJECTIVE: Cystic ameloblastoma, keratocystic odontogenic tumor, dentigerous cyst, and radicular cyst are the most commonly encountered cystic odontogenic lesions. The aim of this study was to investigate the expressions of survivin, E-cadherin, CD138, and CD38 in these lesions and their potential diagnostic usage. MATERIAL AND METHOD: A total of 20 cases, consisting 5 radicular cysts, 5 dentigerous cysts, 5 keratocystic odontogenic tumors and 5 cystic ameloblastomas were included in our series. For all cases, sections from the selected blocks were stained against the antibodies for survivin, E-cadherin, CD138, and CD38 on an automated device. RESULTS: All cystic ameloblastomas and keratocystic odontogenic tumors showed diffuse and strong nuclear survivin expression. No specific survivin immunoreactivity was observed in the dentigerous and radicular cysts. E-cadherin expression was stronger in all dentigerous cysts and radicular cysts when compared to others. CD138 expression in stromal cells was prominent in cystic ameloblastomas, but gradually decreased in the other three lesions. All cases were negative for CD38. CONCLUSION: In the present study, loss of E-cadherin expression in epithelial cells, strong CD138 expression in stromal cells and strong nuclear survivin expression both in epithelial and stromal cells in cystic ameloblastomas and keratocystic odontogenic tumors were the most remarkable findings. These findings are also reinforced by the studies suggesting their role in the aggressiveness and pathogenesis of these tumors.

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The aim of this study was to evaluate the activation status of autophagy in keratocystic odontogenic tumours (KCOT), and to investigate its possible association with growth potential. We detected the expression of some key autophagy-related proteins in clinical samples of KCOT and radicular cysts and compared then by real-time quantitative polymerase chain reaction (qPCR) and immunohistochemical analysis, respectively. The correlation between the autophagy-related proteins tested, and with cell antiapoptotic (Bcl-2) or proliferative (Ki-67) markers in KCOT was explored using Spearman's rank correlation, followed by cluster analysis. The results showed that both the expression of mRNA and the immunoreactivity of the autophagy-related proteins tested were considerably increased in samples of KCOT compared with those in samples of radicular cysts. The correlation analyses showed that the immunostains of autophagy-related proteins in samples of KCOT correlated closely with each other. The immunostains of these autophagy-related proteins also correlated closely with the immunostains of Bcl-2 and Ki-67 in KCOT. More importantly, double-labelling immunofluorescence analyses also showed that the distribution of autophagic and proliferative markers was partially synchronous in samples from KCOT. We have, to our knowledge for the first time, implicated the activation of autophagy in KCOT, and showed its possible association with growth potential.

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OBJECTIVE: In the present study expression of cyclin D1 in the epithelial lining of odontogenic keratocyst, radicular cyst, dentigerous cyst and glandular odontogenic cyst was investigated to compare proliferative activity in these lesions. MATERIAL AND METHOD: Immunohistochemical staining of cyclin D1 on formalin-fixed, paraffin-embedded tissue sections of odontogenic keratocysts (n=23), dentigerous cysts (n=20), radicular cysts (n=20) and glandular odontogenic cysts (n=5) was performed by standard EnVision method. Then, slides were studied to evaluate the following parameters in epithelial lining of cysts: expression, expression pattern, staining intensity and localization of expression. RESULTS: The data analysis showed statistically significant difference in cyclin D1 expression in studied groups (p < 0.001). Assessment of staining intensity and staining pattern showed more strong intensity and focally pattern in odontogenic keratocysts, but difference was not statistically significant among groups respectively (p=0.204, 0.469). Considering expression localization, cyclin D1 positive cells in odontogenic keratocysts and dentigerous cysts were frequently confined in parabasal layer, different from radicular cysts and glandular odontogenic cysts. The difference was statistically significant (p < 0.01). CONCLUSION: Findings showed higher expression of cyclin D1 in parabasal layer of odontogenic keratocyst and the entire cystic epithelium of glandular odontogenic cysts comparing to dentigerous cysts and radicular cysts, implying the possible role of G1-S cell cycle phase disturbances in the aggressiveness of odontogenic keratocyst and glandular odontogenic cyst.

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BACKGROUND: Cytokines were thought to play an important role for the expansion of odontogenic cysts. The purpose of this study was to evaluate the cytokine and chemokine levels of radicular and residual cyst fluids. METHODS: Cyst fluids were aspirated from 21 patients (11 radicular and 10 residual cysts) and the levels of interleukin-1 alpha (IL-1alpha), tumor necrosis factor-alpha (TNF-alpha), monocyte chemotactic protein-1 (MCP-1), and regulated upon activation normal T cell expressed and secreted (RANTES) were determined by ELISA using commercially available kits. RESULTS: Both radicular and residual cyst fluids contained IL-1alpha, TNF-alpha, MCP-1, and RANTES, concentrations of which were significantly higher in the radicular cyst fluids than those in the residual cysts (P < 0.001 for IL-1alpha, TNF-alpha, and RANTES; P < 0.01 for MCP-1). Compared to the other mediators, the concentration of IL-1alpha was found to be highest in both of the cyst fluids. In addition, positive correlations were found between IL-1alpha, TNF-alpha, MCP-1, and RANTES in radicular and residual cyst fluids. CONCLUSION: If the radicular cyst is inadvertently left behind following tooth extraction, some degree of inflammation may carry on. Residual cysts, although to a lesser extend than radicular cysts, have the potential to expand.

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TNF-alpha is a pleiotropic cytokine that is considered as a primary modifier of inflammatory and immune reaction in response to various inflammatory diseases and tumour. We investigated levels of TNF-alpha in 43 radicular cysts and 15 odontogenic keratocysts, obtained from patients undergoing surgery, under local anaesthesia, and after aspiration of cystic fluid from non-ruptured cysts. TNF-alpha is elevated in both cysts' fluid, but higher values were found in radicular cysts in comparison to keratocysts. The significantly higher concentration of TNF-alpha was associated with smaller radicular cysts, higher protein concentration, higher presence of inflammatory cells in peri cystic tissues, and the degree of vascularisation and cysts wall thickness (Mann-Whitney U-test, p < 0.05). No correlation was found based on these parameters in odontogenic keratocyst, but all cysts have detectable concentrations of TNF-alpha. We here for the first time present that a difference in the concentration of TNF-alpha exists between these two cystic types.

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BACKGROUND: The aim of this study was to analyse the usefulness of detecting important apoptosis and proliferation markers in assessing the biological potential of odontogenic keratocysts (OKC) and thus selecting the optimal diagnostic algorithm for these lesions. METHODS: Indirect immunohistochemistry and relevant statistical methods were used for analysis of formalin-fixed and paraffin-embedded samples from 98 patients. RESULTS: Nevoid basal cell carcinoma syndrome (NBCCS) keratocysts were characterized by higher expression of Bcl-2, p27Kip1 and c-erbB-2 as well as by lower proliferative activity measured by Ki-67 in basal cell epithelium and by a lower inflammatory response in comparison with sporadic keratocysts. Dentigerous, radicular and non-specified odontogenic cysts differed from both NBCCS and sporadic keratocysts in a wide spectrum of apoptosis and/or cell cycle-related protein expressions, higher proliferation in the basal cell layer, and vice versa, lower proliferation in the suprabasal cell layer. CONCLUSIONS: The NBCCS keratocysts have a different immunophenotype from sporadic keratocysts and both types are distinguishable from dentigerous, radicular and non-specified odontogenic cysts. These findings confirm the separate biological potential of these lesions and the results of the immunohistochemical analysis have diagnostic and prognostic implications.

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BACKGROUND: Some studies suggest that apoptosis-related factors are involved in the inflammatory processes of marginal periodontal lesions. However, the role of apoptosis in periapical inflammatory lesions remains unclear. We investigated the possible role of apoptotic cell death in periapical inflammatory lesions by means of immunohistochemical analysis of apoptosis-related factors and use of a cell proliferation marker. METHODS: Paraffin-embedded sections of 19 radicular cysts (RCs), and five residual radicular cysts (RRCs) and control specimens of normal gingivae excised from seven cadavers were prepared and examined immunohistochemically with the use of monoclonal antibodies or polyclonal antisera against single-stranded DNA (ssDNA), p53, Bax, Bcl-2, caspase-3, Fas, Fas ligand (Fas-L), and Ki-67 antigen. RESULTS: Epithelium of gingiva, RCs, and RRCs showed expression of ssDNA in suprabasal and superficial epithelial cells and Ki-67 reactivity in basal and parabasal cells. Expression of Ki-67 and ssDNA in RCs and RRCs was slightly higher than that in gingiva. Both Ki-67 and ssDNA reactivity in RCs with intense inflammatory reactions or with thick lining epithelium were significantly stronger than those in RCs with less inflammatory reactions or with thin lining epithelium. Reactivity for p53 was noted sporadically in epithelium of gingiva, RCs, and RRCs, and p53 expression in RCs was significantly greater than that in gingiva. Ki-67 and ssDNA reactivity in RCs increased parallel to the degree of p53 expression. Bax and Bcl-2 were detected in some basal epithelial cells in RCs and RRCs as well as in gingiva. The ssDNA reactivity significantly increased parallel to Bax expression and slightly decreased parallel to Bcl-2 expression in lining epithelium of RCs. Caspase-3 was detected in superficial epithelial cells of both gingiva and lining epithelium of RCs and RRCs, and the distribution of these cells was compatible with the expression of ssDNA. Expression of Ki-67 and ssDNA in caspase-3-positive fields was significantly higher than that in caspase-3-negative fields in RCs. There was very limited expression of Fas and Fas-L in lining epithelium of RCs and RRCs as well as in gingiva. CONCLUSIONS: These data suggest that apoptosis-related factors are involved in the pathophysiologic activity of periapical inflammatory lesions. Such factors may be affected by the structure of lining epithelium and the degree of inflammatory change.

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Platelet-endothelial cell adhesion molecule-1 protein (PECAM-1/CD31) is expressed in numerous physiological and pathological processes characterized by an increase of vascular permeability, and in normal and tumour tissues. CD31, member of the immunoglobulin super-family that mediates cell-to-cell adhesion, is a transmembrane glycoprotein, 130-140 kDa, also know as platelet-endothelium cell adhesion molecule (PECAM-1). CD31 is a ligand for CD38 and plays a role in thrombosis and angiogenesis. CD31 is strongly expressed in endothelial cells and weakly expressed in megakaryocytes, platelets, occasional plasma cells, lymphocytes (marginal zone B-cells, peripheral T-cells) and neutrophils. The present study evaluates the angiogenetic processes which are accompanied by an expansion of cystic radicular and keratocystic lesions of the jaw bone. Twelve subjects with maxillary cysts (8 males and 4 females) with an average age of 43 years were selected by the Chieti University Oral Surgery Department. The surgical samples taken were subjected to histological and immunohistochemical analysis. The histological evaluation confirmed the diagnosis of radicular cystisis and keratocystisis. The immunohistochemical analyses were positive for CD31 protein in all the lesions analysed, even though they had different intensities. Using a semiquantive analysis it was possible to highlight, in the radicular cyst samples, an intense expression of the vascular component both in the inflamed area and the adjacent stroma. The lesions with cheratin content showed newly-formed, rather modest, vascularity both in the area showing slight inflammation, where the cellular component is prevalent, and in the adjacent areas showing no sign of inflammation. Therefore, in our observations, angiogenesis could take on a primary role in the development of cystic lesions of the jaw bones. The differences of CD31 expression, in all samples, would advise for a wider monitoring able to evaluate the possible use of such a protein as a diagnostic marker.

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BACKGROUND: The plasminogen/plasmin proteolytic system participates in a wide variety of extracellular matrix degradation. Detailed knowledge of plasminogen activators (PAs) and their inhibitors may be important for understanding the pathogenesis of radicular cysts. The purpose of this study was to investigate the in situ localization of tissue-type PA (t-PA) and type I PA inhibitor (PAI-1) in radicular cysts. METHODS: Thirty formalin-fixed, paraffin-embedded specimens of radicular cysts were examined using immunohistochemistry. In addition, another section from each radicular cyst specimen was stained with hematoxylin and eosin to assess the presence of inflammatory infiltrates. Differences in t-PA and PAI-1 expression between tissues with low and high levels of inflammation were subsequently analyzed using Fisher's exact test. RESULTS: Both t-PA- and PAI-1-positive cells were detected in the lining epithelium, connective tissue, inflammatory infiltrates, and endothelium. In addition, the t-PA signal was mainly expressed in epithelial cells. However, the PAI-1 signal was mainly expressed in fibroblasts. Moreover, significantly greater t-PA as well as PAI-1 expression was noted in radicular cysts with high levels of inflammation as compared to tissues with low levels of inflammatory cell infiltrates (P < 0.05). CONCLUSIONS: The present study confirms earlier indications of local production of PA and its inhibitor in radicular cysts. In addition, this study further shows the tissue localization of the antigens for t-PA as well as PAI-1, and demonstrates that the expression of both t-PA and PAI-1 increases with the grade of inflammation in radicular cysts.

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Cytokines released by immune system cells play an important role in cyst enlargement. This study aimed to determine, by ELISA, the levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3), and IL-6 in fluid and tissue from human radicular cysts. GM-CSF was found in 42.8% of the fluid samples (164.3 pg/mL) and IL-6 in 92.8% (641.4 pg/mL). No IL-3 was detected in any fluid samples. In the tissue samples, 28.6% were positive for IL-3 (369.2 pg/mL), 86.4% for IL-6 (92.4 pg/mL), and 95.8% for GM-CSF (200.5 pg/mL). It can be concluded that GM-CSF and IL-6 were widely found in the fluid and tissue samples. In contrast, IL-3 was found only in the cystic tissue, even though in few lesions. These cytokines may contribute to the inflammation, cystic growth, and bone resorption that characterize cystic lesions.

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OBJECTIVE: To investigate the immunocytochemical expression of parathyroid-hormone-related protein (PTHrP) in odontogenic jaw cysts. DESIGN: Retrospective study of archival tissue. SETTING: University department, UK. MATERIAL: Odontogenic keratocysts (n=27), and dentigerous and radicular cysts (n=10 each). INTERVENTION: Immunocytochemistry by biotin streptavidin technique. MAIN OUTCOME MEASURE: Intensity of staining of PTHrP determined by TV image analysis. RESULTS: The epithelial linings of all the odontogenic keratocysts, 9/10 dentigerous, and 8/10 radicular cysts showed reactivity for PTHrP mainly localised to the basal and suprabasal layers. Odontogenic keratocyst linings expressed significantly higher levels of PTHrP than those of dentigerous and radicular cysts (P<0.003 in each case). There were no differences in epithelial expression of PTHrP between solitary, recurrent and naevoid basal cell carcinoma syndrome-associated odontogenic keratocysts. The fibrous tissue walls of all types of cyst reacted strongly for PTHrP with a trend towards decreasing intensity from odontogenic keratocysts, to dentigerous and then radicular cysts. CONCLUSION: It is possible that PTHrP modulates growth and bone resorption in odontogenic cysts. PTHrP may act synergistically with interleukin-1 to increase bone resorption or stimulate osteoblasts and inhibit osteoclasts (resulting in reduced resorption) via its transforming growth factor beta-like activity.

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Our work has two main aims: to identify oxygenated sterols that accompany cholesterol in dental cyst and to develop effective methods for "profile" analyses of these sterols. Attention as focused on a family of products derived from cholesterol, characterized by the presence of one or more oxygenated functions. More than fifty of these oxysterols are known and find most of time in different parts of the body. In the procedure, lipids are isolated from dental cyst, the fraction is trimethylsilylated and analysed by capillary gas chromatography. Sterols are identified by comparison with reference compounds. However, two sterols of particular interest, viz cholesterol alpha and beta epoxides, are so easily produced from cholesterol (even when rigorous precautions are taken) that indirect methods of analysis are strongly advisable. An adequate degree of quantification is possible for sterols such as 26-hydroxycholesterol (26OHCL), which do not arise significantly as artefacts. Even to verify the fraction which seems to be 26OHCL we used thin layer chromatography coupled with mass spectrometer.

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The expression of epidermal growth factor receptor (EGFR) was investigated in 67 cases of odontogenic cysts and 35 cases of odontogenic tumors using monoclonal antibody to EGFR (Biomarker, Israel) to determine the presence and significance of this transmembrane growth factor receptor. The cystic epithelial cells of odontogenic cystic lesions (keratocyst 60%; primordial cyst 75%; radicular cyst 35%; and follicular cyst 47.4%) were positive to EGFR staining. Cytochemical characterization of EGFR in those cystic epithelium was cell membrane positive type as in the normal epithelium. No expression of EGFR was found in the odontogenic tumors. This diversity of EGFR represents no binding activity of EGF, or loss of EGFR in the tumor cell upon EGFR mediated growth in odontogenic tumors was suggested a different tumor cell growth factor status or microenvironment in cell proliferation mechanism at the cellular level in cysts and tumors of odontogenic origin.

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The present investigation was undertaken to identify commonly occurring foreign material in postendodontic periapical granulomas and cysts. 29 biopsies from such lesions with observed foreign material were routinely processed, stained with H&E, von Kossa and Calcofluor White and investigated by light, polarization and fluorescence microscopy. Applying back-scattered SEM images, the foreign material was subjected to energy dispersive X-ray analysis. 4 groups of foreign material were observed: 1. Black/brownish fragments and yellow/brown granules containing Au, Ag, Cu, Hg, Sn and Zn compatible with amalgam. 2. Fine black/brown/yellow granules compatible with endodontic sealer components revealing Ag, Ba, Bi, Cu, S, Ti and Zn. 3. Basophilic fragments compatible with Ca salts from Ca(OH), extruded periapically and containing Ca and P. 4. Elongated/rounded/oval/kidney-shaped, colourless to slightly basophilic, birefringent structures revealing C and O and with a slit-like central canal and a bright, pale-blue fluorescence specific for cellulose.

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Human radicular cystic tissue of jaws was found to contain between 0.823 pg/mg to 18.026 pg/mg interleukin 1 beta and from 0.34 pg/mg to 0.708 pg/mg interleukin 1 alpha. No IL-1 beta and alpha could be found in specimens from healthy patients. A finding which may be extremely relevant in cystic growth and episodes of alveolar bone resorption around the cystic lesion.

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The binding sites for Ulex europaeus agglutinin I (UEA-I), Bandeirea simplicifolia agglutinin I (BSA-I), and peanut agglutinin (PNA) were comparatively examined in the surgical materials from 41 cases of cystic and solid ameloblastomas and 42 cases of non-neoplastic odontogenic cysts including dentigerous cyst, odontogenic keratocyst, and radicular cyst. In non-neoplastic cysts, most of epithelial lining layers gave positive binding with UEA-I and BSA-I. However, no positive reactions were obtained for these two lectins in the epithelial components of ameloblastoma, except for limited UEA-I binding to markedly keratinized tumor cells in four cases. PNA binding was irregular and did not make any clear distinction between ameloblastomas and cysts. The results suggest that the lectin staining for UEA-I and BSA-I is a useful histologic aid for differential diagnosis between cystic ameloblastoma and non-neoplastic jaw cysts.

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