Azumi Hirata

Role of osteoclast extracellular signal-regulated kinase (ERK) in cell survival and maintenance of cell polarity.

Morphological changes of osteoclasts by a MEK1 inhibitor, PD98059, were investigated to clarify a role of ERK. PD98059 promoted apoptosis of osteoclasts and the loss of ruffled borders. This study supports the importance of ERK in survival and polarity of osteoclasts.

Localization of Runx2, Osterix, and Osteopontin in Tooth Root Formation in Rat Molars

Cementogenesis starts with the differentiation of cementoblasts. Mature cementoblasts secrete cementum matrix. Cementum components are similar to bone; moreover, cementoblasts possess many characteristics similar to those of osteoblasts. Runx2 and osterix, the transcriptional factors for osteoblast differentiation, participate in tooth formation. However, the characteristics of Runx2 and osterix during the differentiation process of cementoblasts remain unclear. In this study, we examined the immunolocalization patterns of Runx2, osterix, and osteopontin during rat molar tooth formation. Periodontal ligament cells and osteoblasts located on the alveolar bone surface showed immunoreactivity for Runx2. Colocalization of Runx2 and osterix was detected in cementoblasts, which penetrated the ruptured Hertwigs epithelial root sheath and attached to root dentin. Moreover, osteopontin was observed in Runx2-positive cementoblasts facing the root surface. However, the cells adjacent to cementoblasts showed only Runx2 reactivity. Neither Runx2 nor osterix was seen in cementocytes. These results suggest that both Runx2 and osterix are important for differentiation into cementoblasts. Additionally, osterix may be indispensable for transcription of osteopontin expression.

Localization of Perlecan and Heparanase in Hertwig's Epithelial Root Sheath During Root Formation in Mouse Molars

During cementogenesis, dental follicular cells penetrate the ruptured Hertwigs epithelial root sheath (HERS) and differentiate into cementoblasts. Mechanisms involved in basement membrane degradation during this process have not been clarified. Perlecan, a heparan sulfate (HS) proteoglycan, is a component of all basement membranes. Degradation of HS of perlecan by heparanase cleavage affects a variety of biological processes. We elucidated immunolocalization of perlecan and heparanase in developing murine molars to clarify their roles in cementoblast differentiation. At the initial stage of root formation, perlecan immunoreactivity was detected on the basement membrane of HERS. Weak heparanase immunoreactivity was detected in HERS cells. HERS showed intense staining for heparanase as root formation progressed. In contrast, labeling for perlecan disappeared from the basement membrane facing the dental follicle, and weak immunoreactivity for perlecan was detected on the inner side of the basement membrane of HERS. These findings suggest that perlecan removal is an important step for root and periodontal tissue formation. Heparanase secreted by the cells of HERS may contribute to root formation by degrading perlecan in the dental basement membrane.

Localization of Osteoprotegerin (OPG) on Bone Surfaces and Cement Lines in Rat Tibia

Osteoprotegerin (OPG), a soluble member of the tumor necrosis factor (TNF) receptor family, is an osteoclastogenesis inhibitory factor. We investigated the localization of OPG in rat tibia using a specific peptide antibody to clarify the role of OPG in bone remodeling. OPG reactivity was mainly seen on bone surfaces. In bone matrices, OPG was also localized on cartilage/bone interfaces and cement lines. However, labeling was scarcely detected in the region of contact between osteoclasts and stromal cells. Some osteoblasts and osteocytes showed weak labeling. Immunoreactivity was not seen in chondrocytes or osteoclasts. Immunoelectron microscopic observation revealed that OPG is localized on the bone surfaces under osteoclasts. These findings suggest that OPG derived from osteoblast lineage cells and/or serum may be concentrated on resorbed bone surfaces and subsequently on cement lines. OPG may play an important role in the prevention of excess bone resorption by inhibiting differentiation and activity of osteoclasts in bone remodeling.

Primary intraosseous squamous cell carcinomas: Five new clinicopathologic case studies

Primary intraosseous squamous cell carcinomas (PIOSCCs) are rare malignant tumours which arise from odontogenic epithelial remnants. Herein we report five new PIOSCC cases, affecting three female and two male patients with a mean age of 64.4 years. One case involved the maxilla and four cases occurred in the mandible. Typical radiographic findings were ill-defined radiolucencies. Histopathologically, four cases were diagnosed as well-differentiated PIOSCCs arising de novo or from odontogenic cysts. The remaining case was a moderately differentiated de novo PIOSCC. In four cases, treatment consisted of surgical removal with perioperative radiotherapy or chemotherapy. To date, there have been no recurrences or distant metastases in three cases. The lesion was not locally controlled in one case and curative treatments were rejected in another case. PIOSCCs are thought to be important among radiolucent jaw lesions, and early diagnoses and surgical excision with sufficient margins of safety may allow for good prognoses.

Ultracytochemical study of medullary bone calcification in estrogen injected male Japanese quail

Fine structural and cytochemical studies were performed to clarify the pattern of medullary bone calcification, specifically in relation to sulfated glycosaminoglycans, by using estrogen‐induced medullary bone of male Japanese quails. Tibiae were collected at 4 and 7 days after estrogen treatment. Medullary bone had developed inward toward the marrow cavity, and calcification had begun near the cortical bone and deeper parts of the trabeculae, accompanied by wide osteoid at extending tips and surface areas of the trabeculae. Sulfated glycosaminoglycans, detected by high iron diamine (HID), were distributed in the matrix in a pattern similar to that of calcified matrix of the trabeculae. Cortical bone was negatively stained by HID. In undecalcified specimens, calcified nodules were seen in areas undergoing calcification. Globular structures composed of fine filamentous materials, a marginal dense layer, and central core, were also observed in the matrix of decalcified specimens. Both the calcified nodules and globular structures showed the same distribution pattern, i.e., they were dispersed at surface areas and coalesced in the deeper areas of the matrix. The globular structures were exclusively positive for HID–thiocarbohydrazide–silver protein (HID‐TCH‐SP) stain, indicating the localization of sulfated glycosaminoglycans. These results strongly suggest that medullary bone calcification progresses by the coalescence of calcified nodules and that sulfated glycosaminoglycans play an important role for the regulation of globular calcification. Anat Rec 264:25–31, 2001.

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin suggests abnormal palate development after palatal fusion

Mouse embryos exposed to 2,3,7,8‐tetrachloridedibenzo‐p‐dioxin (TCDD) develop cleft palates and hydronephrosis. Cleft palates occur after TCDD exposure due to contact and/or fusion failure. We investigated whether cleft palate can be induced by dissociation of the palatine process after fusion. Pregnant mice on gestational day (GD) 12 were randomly divided into two groups: one group was administered through gastric tubes one dose of olive oil (control group) and the other group was administered one dose of TCDD diluted with olive oil, both at a dose of 40 µg/kg body weight. Embryos were removed by cesarean section from pregnant mice during the palatal formation stage (GD 13–18) and the palatal form was observed using a stereoscopic microscope. In TCDD‐exposed embryos, palatal fusion was observed on GD 14, 15 and 16 and the incidence of cleft palate was 100% on GD 18. Fusion rates were 17.5 ± 15.2% and 12.4 ± 11.8% on GD 15 and 16, respectively. Some palates from the TCDD‐exposed mouse embryos showed clearly developed cleft palate after fusion of the lateral palatine processes during palatal formation. A mass of cells, which were chiefly epithelial in the fused palates was observed in the TCDD‐exposed mouse embryos. A decrease in E‐cadherin expression was observed in this mass of cells, indicating its involvement in the development of cleft palate.

Localization of CD44 (Hyaluronan Receptor) and Hyaluronan in Rat Mandibular Condyle

CD44 is a multifunctional adhesion molecule that binds to hyaluronan (HA), type I collagen, and fibronectin. We investigated localization of CD44 and HA in mandibular condylar cartilage compared with the growth plate and the articular cartilage, to clarify the characteristics of chondrocytes. We also performed Western blotting using a lysate of mandibular condyle. In mandibular condyle, CD44-positive cells were seen in the surface region of the fibrous cell layer and in the proliferative cell layer. Western blotting revealed that the molecular weight of CD44 in condyle was 78 to 86 kD. Intense reactivity for HA was detected on the surface of the condyle and the lacunae of the hypertrophic cell layer. Moderate labeling was seen in cartilage matrix of the proliferative and maturative layer. Weak labeling was also seen in the fibrous cell layer. In growth plate and articular cartilage, HA was detected in all cell layers. However, chondrocytes of these cartilages did not exhibit reactivity for CD44. These results suggest that chondrocytes in the mandibular condylar cartilage differ in expression of CD44 from those in tibial growth plate and articular cartilage. Cell-matrix interaction between CD44 and HA may play an important role in the proliferation of chondrocytes in the mandibular condyle.

The Evaluation of Bone Formation of the Whole-Tissue Periosteum Transplantation in Combination With β-Tricalcium Phosphate (TCP)

We investigated the osteogenic potential of a combination graft of β-tricalcium phosphate (TCP) and periosteum in the rat calvarial defect model. The combination β-TCP and periosteum graft was grafted into rat calvarial defects; the newly formed bone in the defect was studied histologically and radiographically and compared with periosteum grafts and TCP grafts. Ten days after combination grafting, the grafted periosteum showed cell proliferation and Runx2 immunoreaction; 20 days after grafting, new bone formation was seen around the β-TCP; and 30 days after grafting, new bone developed and actively replaced β-TCP, while radiography showed calcified areas. Total bone formation of the combination periosteum and β-TCP graft was significantly increased compared with single grafts of β-TCP or periosteum (P < 0.01). The combination graft of periosteum and β-TCP showed marked bone formation in rat calvarial defects. This result suggests that combination grafts may be effective for repairing bone defects.

Newly Formed Bone Induced by Recombinant Human Bone Morphogenetic Protein-2: A Histological Observation.

Purpose: To observe, histologically, bone induced by recombinant human bone morphogenetic protein-2 (rhBMP-2) in onlay grafted and sinus lifted alveolaris. Material and Methods: Eighteen patients were treated with rhBMP-2 at concentration 1.5 mg/mL with an absorbable collagen sponge (ACS). The treated bone was harvested with small trephine bur at 5 or 7 months after surgery for the micro Computer Scanning (CT) and light microscopic observation. Results: Micro CT showed clearly 3-dimensional trabecular bone structure. New bone formation and bone marrow structure were observed in the observed area. Osteoblastic cells existed along the new bone, and osteopontin was localized in the bone matrix weakly. In the connective tissue around the new bone, many CD34-positive blood vessel cells were present. Some tartrate-resistant acid phosphatase (TRAP)-positive osteoclastic cells were observed around bone at this stage. Conclusion: The application of rhBMP-2 with ACS induced a new bone accompanied by blood vessels in atrophied alveolaris. This suggests that rhBMP-2 is capable of osteoinductivity in human jaw.