Connexin 43 expression in tooth germ and benign odontogenic tumors.

OBJECTIVE: The aim of this study was to investigate and compare the immunohistochemical expression of connexin 43 (Cx43) in tooth germs (TGs), ameloblastic fibromas (AFs), ameloblastic fibro-odontomas (AFOs), and conventional ameloblastomas (AMs). STUDY DESIGN: Nine TGs, 12 AFs, 12 AFOs, and 27 AMs were evaluated for Cx43 expression by immunohistochemistry. RESULTS: Most of the TGs expressed Cx43 in the mesenchyme (77.6%) and in the late stages of odontogenesis. Cx43 was more highly expressed (P < .05) in the mesenchymal layer of all groups than in the epithelial layer except for the AFOs. When comparing the expression of Cx43 in the different layers of the analyzed groups, statistically significant differences were observed between AFO vs AM (*P = .0158) in the epithelial layer and between AF vs AFO (P** = .0046) in the mesenchymal layer. CONCLUSIONS: The results obtained in this study showed that Cx43 is a protein with important expression in the mesenchymal layer of the embryonic and odontogenic tissues studied. It could be speculated that Cx43 participates in mineralization events based on the relationship of the expression of this protein between the epithelial and mesenchymal layers of odontogenic tissues.

Analysis of Protein Immunoexpression and Its Interrelationship in the Pathogenesis of Odontomas and Ameloblastic Fibro-Odontomas: A Systematic Review.

Odontomas and ameloblastic fibro-odontomas (AFOs) are the result of a developmental anomaly of odontogenic tissues. A literature review of proteins immunoexpressed in odontomas and AFOs was conducted in order to determine which proteins are involved in the pathogenesis of these lesions. AFO was changed to early odontoma in the 2017 WHO classification and will also be discussed in this article. A literature search was performed in the following electronic databases: PubMed/MEDLINE, Web of Science, Scopus, EMBASE, Lilacs, Cochrane Collaboration Library, and Science Direct. The research question was developed according to the population, intervention, comparison, and outcome (PICO) framework: Which proteins are related to the differentiation of odontomas and what is their interrelationship with AFOs? Thirty articles met all inclusion criteria and were selected for this systematic review, totaling 355 cases of odontomas and 43 cases of AFO. Similar immunoexpression was observed in odontomas and AFOs. Immunoexpression of proteins involved in cell differentiation was higher in compound odontomas than in complex odontomas. Proteins involved in histodifferentiation and enamel formation were more frequent in odontomas. The immunoexpression of enamel matrix proteins differs between odontomas and tooth germs, with their persistence being related to the development of odontomas. Compound odontomas exhibit the highest immunoexpression of proteins involved in cellular histodifferentiation and the Wnt/beta-catenin pathway is involved in tumor formation.

IKKß overexpression together with a lack of tumour suppressor genes causes ameloblastic odontomas in mice.

Odontogenic tumours are a heterogeneous group of lesions that develop in the oral cavity region and are characterized by the formation of tumoural structures that differentiate as teeth. Due to the diversity of their histopathological characteristics and clinical behaviour, the classification of these tumours is still under debate. Alterations in morphogenesis pathways such as the Hedgehog, MAPK and WNT/ß-catenin pathways are implicated in the formation of odontogenic lesions, but the molecular bases of many of these lesions are still unknown. In this study, we used genetically modified mice to study the role of IKKß (a fundamental regulator of NF-κB activity and many other proteins) in oral epithelial cells and odontogenic tissues. Transgenic mice overexpressing IKKß in oral epithelial cells show a significant increase in immune cells in both the oral epithelia and oral submucosa. They also show changes in the expression of several proteins and miRNAs that are important for cancer development. Interestingly, we found that overactivity of IKKß in oral epithelia and odontogenic tissues, in conjunction with the loss of tumour suppressor proteins (p53, or p16 and p19), leads to the appearance of odontogenic tumours that can be classified as ameloblastic odontomas, sometimes accompanied by foci of secondary ameloblastic carcinomas. These tumours show NF-κB activation and increased ß-catenin activity. These findings may help to elucidate the molecular determinants of odontogenic tumourigenesis and the role of IKKß in the homoeostasis and tumoural transformation of oral and odontogenic epithelia.

Comparison of fatty acid synthase and cyclooxygenase-2 immunoexpression in embryonal, benign, and malignant odontogenic tissues.

OBJECTIVES: The aim of this study was to analyze the immunohistochemical expression of fatty acid synthase (FASN) and cyclooxygenase-2 (COX-2) in tooth germ (TG), ameloblastoma (AM), ameloblastic carcinoma (AC), ameloblastic fibroma (AF), and ameloblastic fibrosarcoma (AFS). STUDY DESIGN: Immunohistochemistry for FASN and COX-2 was performed in 10 TG, 44 AM, 10 AC, 9 AF, and 5 AFS specimens. The results were analyzed by using the immunoreactive score (IRS) and Kruskal-Wallis test followed by Dunn's post-test. RESULTS: Most TG specimens were strongly positive for FASN, whereas COX-2 was weak or negative. All AM and AC specimens expressed both proteins. In AF specimens, FASN and COX-2 were variably expressed in the epithelium and negative in the mesenchyme. In AFS specimens, FASN was strongly positive in the malignant mesenchyme and variable in the epithelium; COX-2 was focal or weak in both components. FASN expression showed significant differences in the following comparisons: TG vs AC, AM vs AC, and AF vs AFS. Differences in COX-2 were significant when comparing TG specimens with AM, AC, and AF specimens. CONCLUSIONS: The results suggest that FASN and COX-2 overexpression may have a role in the pathogenesis of AM and AC, whereas in AFS, FASN seems to be mainly involved. Further studies are necessary to clarify these mechanisms and their clinical implications.

Activated WNT signaling in postnatal SOX2-positive dental stem cells can drive odontoma formation.

In common with most mammals, humans form only two dentitions during their lifetime. Occasionally, supernumerary teeth develop in addition to the normal complement. Odontoma represent a small group of malformations containing calcified dental tissues of both epithelial and mesenchymal origin, with varying levels of organization, including tooth-like structures. The specific cell type responsible for the induction of odontoma, which retains the capacity to re-initiate de novo tooth development in postnatal tissues, is not known. Here we demonstrate that aberrant activation of WNT signaling by expression of a non-degradable form of ß-catenin specifically in SOX2-positive postnatal dental epithelial stem cells is sufficient to generate odontoma containing multiple tooth-like structures complete with all dental tissue layers. Genetic lineage-tracing confirms that odontoma form in a similar manner to normal teeth, derived from both the mutation-sustaining epithelial stem cells and adjacent mesenchymal tissues. Activation of the WNT pathway in embryonic SOX2-positive progenitors results in ectopic expression of secreted signals that promote odontogenesis throughout the oral cavity. Significantly, the inductive potential of epithelial dental stem cells is retained in postnatal tissues, and up-regulation of WNT signaling specifically in these cells is sufficient to promote generation and growth of ectopic malformations faithfully resembling human odontoma.

Pigmented ameloblastic fibro-odontoma: clinical, histological, and immunohistochemical profile.

Ameloblastic fibro-odontoma (AFO) is a slow-growing, expansive, benign odontogenic tumor, composed of ameloblastic epithelium embedded in an ectomesenchymal stroma resembling dental papilla, containing hard dental tissue in variable degrees of maturation, including enamel, dentin, and sometimes cementum. AFO typically affects the posterior mandible, causing bony expansion. We report a case of pigmented AFO in a 5-year-old boy, comprising clinical and histological features illustrated by immunohistochemistry using a large panel of antibodies, polarized light microscopy and scanning electron microscopy.

Immunohistochemical expression of thymosin ß4 in ameloblastomas and odontomas.

Ameloblastoma is regarded to be a benign odontogenic tumor, but it is destructive, locally invasive and presents a high rate of recurrence. Thymosin ß4 (Tß4) is closely associated with tooth germ development. Tß4 also plays a role in malignant progression and invasion. However, little is known about the function of Tß4 in odontogenic tumors. Thus, we investigated Tß4 expression in ameloblastomas and compared it with odontomas. We immunohistochemically evaluated the expression of Tß4, ameloblastin (AMBN), amelogenin (AMEL) and enamelin (ENAM) in 57 samples of ameloblastomas from 40 patients, and also assessed the expression of these molecules in 11 cases of odontomas, two of ameloblastic fibro-odontomas and one of tooth germ-like structures without the formation of enamel and dentin. Tß4 signals were observed in almost all of the ameloblastomas. The signals were observed in both peripheral columnar cells and central polyhedral/angular cells. Similar findings were observed in tooth germ-like structures, and in the ameloblastomatous nests in the ameloblastic fibro-odontomas. These samples had negative results for AMBN, AMEL and ENAM. Meanwhile, Tß4 signals were not seen in the odontomas, although immunolabeling for AMBN, AMEL and ENAM was observed in the enamel matrix and in some ameloblasts. Ectomesenhymal regions in the odontomas were negative for staining with the antibodies for AMBN, AMEL and ENAM. These results suggest that Tß4 could be associated with morphogenesis and tumor invasion in the ameloblastoma, and that Tß4 may play a role in the behavior of ameloblastoma.

Expression of cytokeratins in the epithelium of canine odontogenic tumours.

Odontogenic tumours are considered to be relatively rare; however, several histologically distinct types have been identified in dogs. The more common canine odontogenic tumours are peripheral odontogenic fibroma and canine acanthomatous ameloblastoma. The expression of cytokeratins (CKs) has been established for the human dental germ and odontogenic tumours. The aim of the present study was to describe the immunohistochemical expression of a panel of CKs in the epithelium of the canine dental germ, normal gingiva and odontogenic tumours arising in this species. Samples from 20 odontogenic tumours, 12 tooth germs and three normal gingival tissues were obtained. Each sample was stained with haematoxylin and eosin and subjected to immunohistochemistry for CK expression. The typical expression pattern of CKs in the odontogenic epithelium and gingiva of dogs was CK14 and CK5/6. CKs 7, 8, 18 and 20 were generally absent from the canine dental germ, gingiva and odontogenic tumours. Dogs and man therefore exhibit similar CK expression in the odontogenic epithelium.

Podoplanin expression in odontomas: clinicopathological study and immunohistochemical analysis of 86 cases.

Podoplanin, a sialomucin-like transmembrane glycoprotein, is currently used as a specific marker for lymphatic vessels. However, podoplanin expression has also been linked to tooth development. To investigate the expression of podoplanin in odontomas, 86 tissue samples were classified and then analyzed using immunohistochemical methods. Formalin-fixed, paraffin-embedded specimens were collected and classified, followed by immunohistochemical examination. The majority of the odontomas (66.3%) were the compound type, and the remainder (33.7%) were the complex type. The patients ranged in age from 2 to 89 years (mean, 23.9 years), and 45 (52.3%) of them were male and 41 (47.7%) were female. The most common location for complex odontomas was the molar region of the mandibular bone, and that for compound odontomas was the maxillary incisor region. Immunohistochemistry revealed that developing and mature odontoblasts, Tomes' fibers, and pulp cells near podoplanin-positive odontoblasts were positive for podoplanin. In addition, podoplanin positivity was evident in secretory ameloblasts, but not in mature ameloblasts. The pattern of podoplanin expression in odontomas corresponds to development of the tooth germ, and appears to be influenced by the stage of differentiation of the lesion, suggesting that the protein may participate in the process of differentiation.

Aspiration cytology of ameloblastic fibroma: a diagnostic challenge.

Ameloblastic fibroma of the jaw is a rare, benign mixed odontogenic tumor, having little tendency for local invasion and a low recurrence rate. Cytologic distinction from ameloblastoma, ameloblastic fibrosarcoma, and intraosseous adenoid cystic carcinoma is necessary, in view of the different biologic behavior. A painful, slow-growing swelling of the jaw in a 5-yr-old child clinicoradiologically considered as a benign cystic lesion was aspirated. Sheets of small monomorphic epithelial cells with peripheral palisading by columnar cells were seen on cytology smears. The striking feature was central hyaline globules in some tubules. A cytologic possibility of adenomatoid odontogenic tumor was suggested. Histopathology, however, confirmed it to be an ameloblastic fibroma.

The secretion of amelogenins is associated with the induction of enamel and dentinoid in an ameloblastic fibro-odontoma.

Ameloblastic fibro-odontoma is the unique entity of epithelial-ectomesenchymal odontogenic tumors, which is characterized by enamel formation in addition to dentine. We examined immunohistochemically a case of this tumor in which enamel having prism structures was developed in the absence of odontoblast differentiation but was in contact with mesenchymal matrices. Histological examination showed diverse morphological features of epithelial tumor cells, e.g., cuboidal cells comprising tooth bud-like projections, ameloblast- and stellate reticulum-like cells, and residual cells in forms of extended cords or islands of odontogenic epithelium. Immunostaining with anti-amelogenin sera proved that the intracellular production of amelogenins was initiated at the tooth bud-like stage. The secreted amelogenins were detected almost exclusively in the induced enamel and dentinoid areas, as well as in the core region of cementicle-like spheres deposited in the encapsulating stroma. The results obtained indicate that the odontogenic tumor epithelia and its products, i.e., amelogenins, participate in multifaceted aspects of dental hard tissue formation that takes place during oncogenesis.

Immunohistochemical expression of neural tissue markers (neuron-specific enolase, glial fibrillary acidic protein, S100 protein) in ameloblastic fibrodentinoma: a comparative study with ameloblastic fibroma.

Formalin-fixed paraffin-embedded sections of three cases of ameloblastic fibrodentinoma (AFD) were studied by the avidin-biotin-peroxidase complex method using antibodies against neuron-specific enolase (NSE), glial fibrillary acidic protein (GFAP) and S100 protein and the results were compared with those in ameloblastic fibroma (AF). A striking histopathological characteristic of AFD was the formation of abortive dentin with various degrees of maturation at the epithelial-mesenchymal tissue interface. Central cells of enamel organ-like epithelia with various stages of abortive dentin induction in AFD were generally positive for NSE. Dental lamina-like epithelial cells also showed positive staining in some areas. No cells were positive for NSE in AF. Positive staining for GFAP was observed in the juxta-epithelial mesenchymal tissue of the formation stage of immature dentin with various numbers of entrapped cells in AFD, but GFAP staining was negative in other mesenchymal and epithelial tissues at other stages. In AF, no GFAP-positive cells were found. There were a few S100 protein-positive cells found in the foci of epithelial components in both AFD and AF. Mesenchymal cells showing a dendritic or spindle shape were positive for S100 protein in some areas of AFD and AF. Although such cells in the mesenchymal component of pigmented AFD were more numerous than in non-pigmented AFD and AF, their distribution pattern in the former condition was basically similar to that in the latter. Although the present results, obtained from conventional immunohistochemical procedures, do not directly reflect the expression of neural crest-derived cells in the dentinogenesis of AFD, such results do not disprove the possibility of the expression of neural proteins probably related to neural crest-derived cells in dentinogenesis under certain pathologic conditions in odontogenic mixed tumors. Such a phenomenon may also occur during dentinogenesis in other odontogenic mixed tumors and in normal tooth differentiation, but at an undetectable level.

Immunohistochemical demonstration of an enamel sheath protein, sheathlin, in odontogenic tumors.

Enamel proteins can be useful markers for assessment of the functional differentiation of neoplastic epithelium and the nature of extracellular matrices in odontogenic tumors. In the present study, we examined immunohistochemical localization of sheathlin, a recently cloned enamel sheath protein, in various odontogenic tumors to evaluate functional differentiation of tumor cells and the nature of hyalinous or calcified matrices in odontogenic neoplasms. Distinct immunolocalization of sheathlin was observed in the immature enamel of the tooth germ at the late bell stage. Secretory ameloblasts facing the enamel matrix also showed positive staining in their cytoplasm. Definite localization of sheathlin was demonstrated in the enamel matrix in odontogenic tumors with inductive dental hard tissue formation such as ameloblastic fibroodontomas and odontomas. Immunoexpression of sheathlin was, furthermore, demonstrated in eosinophilic droplets in solid nests of adenomatoid odontogenic tumor (AOT) and ghost cells in the epithelial lining of calcifying odontogenic cyst (COC). In AOT, cells facing the eosinophilic droplets also expressed the protein in their cytoplasm. There was neither intracellular staining for sheathlin in the tumor cells nor extracellular staining in the matrix of ameloblastomas and calcifying epithelial odontogenic tumors. Dentin, dysplastic dentin-like hyaline material and cementum in the tumors examined were negative for sheathlin. These results show that immunodetection of sheathlin is a useful marker for functional differentiation of secretory ameloblasts and enamel matrix, which is often hard to differentiate from other hard tissues in odontogenic tumors. Our findings from the view point of sheathlin expression support that the tumor cells of ameloblastomas do not attain full differentiation into functional ameloblasts. It is very interesting that epithelial cells in odontogenic tumors can differentiate into functional ameloblasts without induction by odontogenic mesenchyme, as shown by immunoexpression of sheathlin in eosinophilic droplets within solid epithelial sheets in AOT and ghost cells in the epithelial lining of COC where inductive participation of mesenchymal cells was most unlikely.

[Ameloblastic fibrosarcoma: an immunohistochemical and ultrastructural study of the mesenchymal component].

To study the characteristics of the mesenchymal cells of ameloblastic fibrosarcoma (AFS), three cases of AFS were studied immunohistochemically and ultrastructurally. The results showed that the mesenchymal component of AFS consisted predominantly of fibroblastic cells with a small number of undifferentiated cells, a few histiocytes and occasionally myofibroblastic cells under electron microscope. The fibroblastic cells were Vimentin positive only, and myofibroblastic cells were positive for Vimentin, HHF35 and alpha-SMA. The histiocytes were positive both for kp1 and PG-M1, suggesting that these cells were infiltrating cells from peripheral blood rather than histiocytic differentiation of tumor cells. Compared with ameloblastic fibroma, AFS showed much higher PCNA labeling index, suggesting higher proliferation activity of AFS.

[An immunohistochemical study of the proliferating activity of ameloblastic fibroma and ameloblastic fibrosarcoma].

Six cases of ameloblastic fibroma (AF) and three cases of ameloblastic fibrosarcoma (AFS) were immunohistochemically investigated with a proliferating cell nuclear antigen (PCNA) monoclone antibody. Data on the PCNA labeling index (LI) of both epithelial and mesenchymal components were analyzed. The results showed that the PCNA LI of mesenchymal component of AFS (40.8%) was significantly higher than that of AF (3.2%) (P < 0.01) and that of the epithelial component of AFS (5.3%) was significantly lower than that of the mesenchymal one of AFS (P < 0.01). The results suggested that PCNA LI be a useful marker for the differentiation diagnosis of AF and AFS.

Calcifying odontogenic cyst associated with compound odontoma: a study on undemineralized material.

In a minority of cases of calcifying odontogenic cyst (COC) it is possible to observe the formation of dental hard tissues in the cyst wall. The use of undemineralized sections has allowed an evaluation of the mineralized tissues normally lost with the use of demineralizing agents. All the dental hard tissues presented a high degree of morpho- and histodifferentiation. The histochemical staining for calcium salts (von Kossa) showed the presence of areas of low mineralization in the portion of the lesion, where the tissue maturation was not complete. In conclusion the appearance of the dental hard tissues in this case of compound odontoma arising in the cyst wall of a COC is similar to that already described in compound odontoma not associated with COC.

Immunohistochemical expression of amelogenins in odontogenic epithelial tumours and cysts.

Amelogenins, enamel proteins in odontogenic tumours, were detected immunohistochemically using a monoclonal antibody. They were strongly expressed in amyloid-like material, ghost cells, and the cells surrounding ghost cells of calcifying epithelial odontogenic tumours and cysts, whereas calcified bodies within the tumours and cysts showed negative staining. The expression of amelogenins was also positive in tumour cells of ameloblastoma, adenomatoid odontogenic tumour, squamous odontogenic tumour and ameloblastic fibroma. Peripheral tumour cells of the follicular ameloblastoma were positive with relatively intense staining. Undifferentiated or flattened tumour cells of adenomatoid odontogenic tumour and non-keratinized tumour cells of the squamous odontogenic tumour showed marked staining. Reduced ameloblasts in the odontoma displayed the strongest staining for amelogenins. The study suggests that biosynthesis of amelogenins may occur in the homogeneous materials of calcifying epithelial odontogenic tumours and cysts.

Melanin pigment in ghost cells of a complex odontoma.

A case of complex odontoma, which contained melanin pigment in the ghost cells in a 53-year-old Japanese woman is reported. In addition to the characteristic histopathologic features of complex odontoma, ghost cells containing melanin pigment were widely distributed in and around the calcified masses. Furthermore, cytoplasmic processes of dendritic cells (melanocytes) were seen in some parts. The possible histogenesis of melanin pigment in odontogenic lesions was discussed, although no conclusion could be drawn from the present study as to its origin in complex odontoma.

Keratinization in odontogenic tumors.

The potential of odontogenic epithelium to keratinize in the form of ghost cells is demonstrated in the histologic variants of a number of odontongic tumors. Although the cells lack keratohyaline granules, they do contain abundant tonofilaments and probably represent an altered form of keratin. The presence of this material in odontogenic tumors does not appear to alter clinical occurence or clinical behavior.