Yoshifumi Tajima

Establishment and characterization of a culture system for enzymatically released rat dental pulp cells.

Abstract. To establish a cell culture system that reflects the dentin formation in dental pulp tissue, we used dental pulp cells enzymatically isolated from rat incisor teeth. During the 20-day culture period, the cells exhibited various phenotypes of the odontoblast differentiation process, from the immature stage to the terminal mineralization stage. The cells began to form the mineralized nodules from day 10, and the nodules became larger by day 20. Alkaline phosphatase (ALP)-positive cells surrounded the mineralized nodules. The ALP activity in the cell layers was maximal on day 5, and gradually decreased to day 20. The calcium content in the cell layers was very low by day 10, and significantly increased from day 15. Sulfated glycosaminoglycans (GAGs) contained in the cell layers increased by day 15, but the content then decreased by day 20. The dental pulp cells produced a small amount of osteocalcin that was released into the culture medium by day 10, and the amount secreted increased markedly from day 15. The expression of osteocalcin and parathyroid hormone/parathyroid hormone-related peptide (PTH/PTHrP) receptor mRNA was evident as early as day 5, and the expression of each gradually increased with the number of days in culture. Dentin matrix protein (Dmp1) mRNA gene transcripts were identified by use of the reverse transcription polymerase chain reaction (RT-PCR) in the cells throughout the culture period. The present results demonstrate that this culture system is useful for studying the differentiation process of the odontoblast-like cells.

Odontogenic cyst giving rise to an adenomatoid odontogenic tumor: report of a case with peculiar features.

Abstract It has been reported that some odontogenic cysts occur in association with odontogenic tumors. 1 Because neoplastic and hamartomatous abberations can occur at any stage of odontogenesis, 2 combined features of odontogenic tumors with epithelial and mesenchymal components may arise within odontogenic cysts. The authors report an unusual example of an odontogenic cyst that contained focal areas of odontogenic epithelial and mesenchymal proliferation exhibiting a combination of adenomatoid odontogenic tumor (AOT) and dentinoid components. Emphasis is placed on the clinical, radiographic, and microscopic features of this case.

Interaction of SNARE proteins in rat parotid acinar cells.

The protein-protein interaction between [soluble NSF attachment protein (SNAP) receptor] (SNARE) proteins found in the lysate of parotid acinar cells was investigated. Immunoblotting analysis showed that parotid acini contain both syntaxin-4 and SNAP-23, plausible candidates of target membranes (t-) SNAREs in non-neuronal cells. However, when vesicle-associated membrane protein (VAMP)-2 was immunoprecipitated from lysates of parotid acinar cells, syntaxin-4 and SNAP-23 were not coprecipitated with VAMP-2, although syntaxin-1 and SNAP-25, t-SNAREs in neuronal cells, were clearly coprecipitated with VAMP-2 from brain lysates. Inversely, when syntaxin-4 was immunoprecipitated from parotid lysates, SNAP-23, Munc18c, and N-ethylmaleimide-sensitive fusion protein (NSF) were coprecipitated, but VAMP-2 was again undetectable. When proteins in the crude secretory-granule fraction were biotinylated and then immunoprecipitated with anti-VAMP-2, 35- and 80-kDa proteins were coprecipitated along with VAMP-2. These results suggest that the interaction between syntaxin-4, SNAP-23 and VAMP-2 is fairly weak and their concentrations in the cell lysate are insufficient to make a readily detectable complex, and that bindings between these proteins are hindered by other proteins in parotid acinar cells.

Ameloblastoma arising in calcifying odontogenic cyst: Report of a case

An unusual case of an ameloblastoma with adenoid-cribriform features arising in calcifying odontogenic cyst is reported. A possible relationship between this peculiar variant of calcifying odontogenic cyst and a conventional ameloblastoma is discussed.

Calcifying odontogenic cyst immunohistochemical detection of keratin and involucrin in cyst wall.

Calcifying odontogenic cysts (COC) were immunohistochemically described using different keratin proteins and involucrin as well as histopathology. The cystic lining epithelium was composed of calcifying, keratinizing, squamous, and columnar epithelial cells, and included calcified masses of irregular shape and various size as well as ghost cells. Calcifying epithelium gave negative or only trace staining for keratins detected with low molecular keratin (PKK1), but were regularly positive with high molecular keratin (KL1) and polyclonal antibody for keratin (TK). They were occasionally positive for involucrin. The cells located in the periphery of the calcified masses had a particular abundance of high molecular weight and total keratins (KL1 and TK). Calcified bodies and ghost cells were devoid of any immunoreactivity. Squamous epithelium was relatively similar to that of normal squamous cell epithelium in the oral mucosa. It were most commonly found in columnar cystic epithelial cells which displayed intense staining with all immunoreagents. It is postulated that such epithelial cells may have a strong potentiality to transform into ghost cells or to undergo metaplasia. They may develop altered synthesis of homogenous acellular materials and finally become transformed into calcifying epithelium containing dystrophic calcified masses.

Comparison of proliferating cell nuclear antigen expression in odontogenic keratocyst and ameloblastoma: an immunohistochemical study

Proliferating cell nuclear antigen (PCNA) is a nuclear protein synthesized in the late G1 and S phase of the cell cycle, and immunohistochemical detection of the protein represents a useful marker for the proliferating fraction of cells in tissue specimens. PCNA expression was studied in odontogenic keratocysts (n = 15) and ameloblastomas (n = 46) using an avidin–biotin–peroxidase complex method on routinely processed paraffin sections. The percentage of PCNA-positive cells determined by point counting was significantly lower in the ameloblastomas (mean 9.4%, standard deviation (SD) 11.0) than in odontogenic keratocysts (mean 29.9%, SD 24.0). In ameloblastomas, the mean percentage of PCNA-positive cells was lowest in the acanthomatous pattern and highest in plexiform pattern. The mean percentage of PCNA-positive cells in plexiform pattern was non-significantly higher than that in follicular pattern. The mean percentage of PCNA-positive cells in plexiform and follicular patterns was significantly higher than that in cyctic and acanthomatous patterns. The frequency of PCNA-positive cells was significantly higher in the peripheral cells of follicular and plexiform patterns than in the central cells of both patterns (p < 0.01). Therefore, peripheral cells were regarded as reserve cell of central cells. The mean percentage of PCNA-positive cells in the epithelial lining of odontogenic keratocyst was not significantly different from those in the peripheral cells of follicular and plexiform patterns of ameloblastoma. In contrast, the odontogenic keratocyst exhibited a mean percentage of PCNA-positive cells which was statistically higher than that in other histological elements of ameloblastomas. The present study suggests that odontogenic keratocyst is regarded as benign odontogenic tumour.

Immunohistochemical profile of basic fibroblast growth factor and heparan sulphate in adult rat mandibular condylar cartilage.

Basic fibroblast growth factor (bFGF) and heparan sulphate (HS) were detected immunohistochemically in mandibular condylar cartilage, and the findings compared with those on epiphyseal articular cartilage. In the condylar cartilage, both bFGF and HS were localized in chondrocytes throughout the various zones including the fibrous, proliferative, mature-cell and hypertrophic zones: bFGF immunostaining was most significant in the proliferative and mature-cell zones, while intense staining for HS was found mainly in the hypertrophic zone. Immunoreaction for bFGF was detected in the nuclei of chondrocytes, whereas HS staining was observed in the cytoplasm. In articular cartilage, only chondrocytes beneath the superficial zone (intermediate zone) demonstrated both bFGF and HS immunoreactivities. Chondrocytes in the deeper calcifying region of the articular cartilage did not immunoreact for either bFGF or HS. These findings suggest that, in contrast to the epiphyseal articular cartilage, a continuous bFGF-mediated remodelling of cells and matrix takes place in mandibular condylar cartilage during the process of endochondral ossification.

SNAP-23 is not essential for constitutive exocytosis in HeLa cells.

We applied the small interfering RNA (siRNA) technique and over‐expression of a dominant‐negative mutant to evaluate the role of SNAP‐23, a non‐neuronal isoform of SNAP‐25, in constitutive exocytosis from HeLa cells. Although the protein level of SNAP‐23 was reduced to less than 10% of the control value by siRNA directed against SNAP‐23, exocytosis of SEAP (secreted alkaline phosphatase) was normal. Double knockdown of SNAP‐23 and syntaxin‐4 also failed to inhibit the secretion. Furthermore, over‐expression of δC8‐SNAP‐23, a dominant‐negative mutant of SNAP‐23, did not abrogate SEAP secretion. These results suggest that SNAP‐23 is not essential for constitutive exocytosis of SEAP.

Ovariectomy Causes Cell Proliferation and Matrix Synthesis in the Growth Plate Cartilage of the Adult Rat

The in vivo effects of ovariectomy in rats have been studied on cell proliferation and matrix synthesis in the growth plate cartilage by assessing immunohistochemically the levels of proliferating cell nuclear antigen and chondroitin sulphate proteoglycan(s). The serum levels of insulin-like growth factor-I and growth hormone were also measured by radioimmunoassay procedures. At 5 weeks after ovariectomy, the serum levels of the growth factor were significantly higher than those in sham-operated rats. In contrast, the level of growth hormone was lower. The nuclear staining of proliferating cell nuclear antigen was generally seen in the zone of proliferative chondrocytes from both groups of rats. Whereas almost all chondrocytes in the proliferative zone of ovariectomized rats expressed proliferating cell nuclear antigen immunoreactivity, fewer did so in that of the sham rats. Quantitative image analysis by ACAS 570 laser cytometry demonstrated that the n uclear antigen-positive sites in ovariectomized rats had significantly higher integrated values (staining intensity), areas and perimeters than those in sham rats. In addition, the number of chondroitin sulphate proteoglycan-immunoreactive cells in the proliferative chondrocytes was also higher in ovariectomized rats than in sham ones. These results suggest that ovariectomy significantly stimulates the cell proliferation and matrix synthesis in the growth plate cartilage, probably through the higher serum level of insulin-like growth factor-I.

AMELOBLASTIC FIBROSARCOMA ARISING DE NOVO IN THE MAXILLA

An ameloblastlc fibrosarcoma (AFS) arlsing in the maxilla of a 14‐yearold male Is descrlbed. The tumor orlglnated from the alveolar bone of the right maxllia with no apparent history of pre‐exlstlng leslon. Histologically, the lesion was composed of benign‐appearlng epithelial Islands and strands scattered within an exceedingly cellular mass of mesenchymet tissue comprislng 8 large number of steliate‐and spindle‐shaped fibroblast‐like cells with marked pleomorphlsm. Occasional cementum‐like calcification was also noted. Immunohistochemically, the neoplastlc mesenchymal cells were positive only for vimentln, whereas the ameioblast‐like epithelial component showed a dlstlnctly positive reactlon for wide‐spectrum keratin and squamous cytokeratin. Clinicopathological features of the current case, as well as previously reported examples of AFS originating from the maxilla, are briefly discussed.