Naoto Suda

Participation of oxidative stress in the process of osteoclast differentiation.

In the present paper, the involvement of active oxygen species in bone resorption has been studied. In order to compare the production of active oxygen by mouse marrow culture cells, fluorescence due to peroxides reacted with 2,7-dichlorofluorescin was measured. After marrow cells were cultured with 1,25-(OH)2D3 for 8 days, there were tartrate resistant acid phosphatase positive multinucleated cells (TRACP(+)MNCs), TRACP positive mononucleated cells, macrophage-like cells and marrow derived stromal cells. Among these cells, TRACP(+) cells could produce almost the equivalent amount of peroxides as could the macrophage-like cells. In order to examine the role of active oxygen in bone metabolism, the amount of oxidative stress was altered during the culture period in the same marrow culture system. Catalase, a catabolic enzyme of hydrogen peroxide (H2O2), significantly suppressed the formation of TRACP(+)MNCs in a dose dependent manner. This suppression was limited in the early stage of the culture period and was reduced by the addition of exogenous H2O2 to culture. Moreover, when superoxide dismutase, a converting enzyme from superoxide anion to H2O2, was added in this system, the formation of TRACP(+)MNCs was significantly increased. These results strongly suggest that active oxygen species, especially H2O2, may be involved in the regulation of osteoclast formation.

The control of vascular endothelial cell injury

The mechanism by which MCI-186 showed a potent cytoprotective effect on the in vitro endothelial cell injury due to 15-HPETE was studied. Stimulation of human leukocytes with various chemical mediators such as TPA, f-Met-Leu-Phe, LTB4, etc. elicited the production of active oxygens, which could be detected by luminol-dependent chemiluminescence. Among the chemical mediators tested, TPA elicited the chemiluminescence the most, and f-Met-Leu-Phe and LTB4 came next. When the leukocytes were directly placed on a monolayer of cultured endothelial cells, followed by stimulating the leukocytes with TPA, severe endothelial cell injury was observed. The effect of TPA was dose dependent. There was good correlation between the active oxygen releasing activity and the cytotoxic activity. When the leukocytes were placed on a filter which was set apart from the monolayer of endothelial cell in a culture dish, and stimulated the leukocytes with TPA, no cytotoxicity was observed. These data strongly suggest that the substance responsible for the cytotoxicity must be a very labile and short-lived substance, presumably active oxygens. On the other hand, MCI-186 was found to have a complete quenching activity to the chemiluminescence due to active oxygens in the TPA-leukocyte system. Taken together, these factors indicate that the potent cytoprotective effect of MCI-186 may be due to its specific radical scavenging activity.

An in situ hybridization study of Runx2, Osterix, and Sox9 at the onset of condylar cartilage formation in fetal mouse mandible

Mandibular condylar cartilage is the principal secondary cartilage, differing from primary cartilage in its rapid differentiation from progenitor cells (preosteoblasts/skeletoblasts) to hypertrophic chondrocytes. The expression of three transcription factors related to bone and cartilage formation, namely Runx2, Osterix and Sox9, was investigated at the onset of mouse mandibular condylar cartilage formation by in situ hybridization. Messenger RNAs for these three molecules were expressed in the condylar anlage, consisting of preosteoblasts/skeletoblasts, at embryonic day (E)14. Hypertrophic chondrocytes appeared at E15 as soon as cartilage tissue appeared. Runx2 mRNA was expressed in the embryonic zone at the posterior position of the newly formed cartilage, in the bone collar and in the newly formed cartilage, but expression intensity in the newly formed cartilage was slightly weaker. Osterix mRNA was also expressed in the embryonic zone and in the bone collar, but was at markedly lower levels in the newly formed cartilage. Sox9 mRNA was continuously expressed from the embryonic zone to the newly formed cartilage. At this stage, Sox5 mRNA was expressed only in the newly formed cartilage. These results suggest that reduced expression of Osterix in combination with Sox9–Sox5 expression is important for the onset of condylar (secondary) cartilage formation.

Parathyroid Hormone-Related Protein Regulates Proliferation of Condylar Hypertrophic Chondrocytes

The condylar cartilage, an important growth site in the mandible, shows characteristic modes of growth and differentiation, e.g., it shows delayed appearance in development relative to the limb bud cartilage, originates from the periosteum rather than from undifferentiated mesenchymal cells, and shows rapid differentiation into hypertrophic chondrocytes as opposed to the epiphyseal growth plate cartilage, which has resting and proliferative zones. Recently, attention has been focused on the role of parathyroid hormone–related protein (PTHrP) in modulating the proliferation and differentiation of chondrocytes. To investigate further the characteristic modes of growth and differentiation of this cartilage, we used mice with a disrupted PTHrP allele. Immunolocalization of type X collagen, the extracellular matrix specifically expressed by hypertrophic chondrocytes, was greatly reduced in the condylar cartilage of homozygous PTHrP‐knockout mice compared with wild‐type mice. In contrast, immunolocalization of type X collagen of the tibial cartilage did not differ. In wild‐type mice, proliferative chondrocytes were mainly located in both the flattened cell layer and hypertrophic cell layer of the condylar cartilage, but were limited to the proliferative zone of the tibial cartilage. The number of proliferative chondrocytes was greatly reduced in both cartilages of homozygous PTHrP‐knockout mice. Moreover, apoptotic chondrocytes were scarcely observed in the condylar hypertrophic cell layer, whereas a number of apoptotic chondrocytes were found in the tibial hypertrophic zone. Expression of the type I PTH/PTHrP receptor was localized in the flattened cell layer and hypertrophic cell layer of the condylar cartilage, but was absent from the tibial hypertrophic chondrocytes. It is therefore concluded that, unlike tibial hypertrophic chondrocytes, condylar hypertrophic chondrocytes have proliferative activity in the late embryonic stage, and PTHrP plays a pivotal role in regulating the proliferative capacity and differentiation of these cells.

Disturbed tooth development in parathyroid hormone-related protein (PTHrP)-gene knockout mice

Parathyroid hormone-related protein (PTHrP) is involved in epithelial-mesenchymal cell interactions during development of various tissues and organs. Tooth germ development is a classical model for this interaction. In tooth germs, PTHrP is expressed in the enamel organ (epithelial component), whereas its major receptor, the type I PTH/PTHrP receptor is expressed in cells of the alveolar bone and dental follicle (mesenchymal components). To clarify the role of PTHrP during fetal tooth germ development, PTHrP gene-knockout mice were used for histochemical and ultrastructural analysis. In wild-type mice, osteoclastic cells were aligned predominantly in the inner aspects of the alveolar bone surrounding the developing tooth germs throughout the late embryonic (after embryonic, 17.5 days) and neonatal animals examined. In contrast, osteoblasts were predominant in corresponding areas of fetal homozygous PTHrP-gene knockout mice with only occasional osteoclasts. In such areas, cell-free surfaces showing cement line-like tartrate-resistant acid phosphatase (TRAP) reactions were frequently observed. In neonatal homozygous mice, bone spicules were often shown to penetrate and/or compress the enamel organ and caused partial destruction of the tooth germs. Osteoclasts were few in number in the inner aspects of the alveolar bone, and had poorly developed ruffled border. No morphological abnormality was noted in cells of the tooth germs proper. On bone surfaces away from developing tooth germs, functional osteoclasts with structural features similar to those in wild-type mice were observed in homozygous mice. These observations suggest that PTHrP is required to maintain an appropriate spatiotemporal arrangement of bone cells and osteoclast function, which are necessary for the normal development of tooth germ and alveolar bone encasing the tooth germ. The observation also demonstrates that PTHrP deficiency affects the structure and function of osteoclasts exclusively those located in the vicinity of the growing tooth germ.

Effects of Maxillary Protraction on Craniofacial Structures and Upper-Airway Dimension

This study was conducted to examine the effect of treatment with a maxillary protraction appliance on the development of the craniofacial structures and upper-airway dimensions. A total of 25 patients (mean age: 9.8 years) with Class III malocclusions were evaluated by the use of lateral cephalograms. A significant increase in maxillary forward growth, inhibition of mandibular forward growth, and clockwise rotation of the mandible were observed. The maxillary incisors were significantly proclined and the mandibular incisors significantly retroclined. A multiple-regression analysis revealed that maxillary growth had a significant positive effect on the superior upper-airway dimension. These findings indicate that the superior upper-airway dimension can be altered during maxillary protraction.

Runx2-deficient mice lack mandibular condylar cartilage and have deformed Meckel's cartilage.

Runx2 (runt-related transcription factor 2) deficient mice lacked the mandibular condylar cartilage and the mandibular bone. The anlage of the condylar process consisted of mesenchymal condensation, which expressed Type I collagen mRNA and alkaline phosphatase activity, but not Type II collagen and aggrecan mRNAs. Therefore, the differentiation of the mandibular condylar cartilage stopped at the preosteoblast (skeletoblast) stage. The lateral pterygoid muscle was attached to this anlage, and relatively abundant mesenchymal condensations were also formed at the muscle-attaching sites, e.g. the anlage of the mandibular body, the angular and coronoid processes. Three-dimensional reconstruction models showed that each mesenchymal condensation was connected to one another, and roughly outlined the shape of the mandible. Meckel’s cartilage in the Runx2-deficient mice had two ectopic cartilaginous processes to which the digastric and myohyoid muscles were attached. These findings indicate that Runx2 is essential for the formation of the mandibular condylar cartilage, as well as for normal development of Meckel’s cartilage and that muscle tissues influence mandible morphology.

ADAMTSL6β protein rescues fibrillin-1 microfibril disorder in a Marfan syndrome mouse model through the promotion of fibrillin-1 assembly.

Background: The pathology of Marfan syndrome is caused by insufficient fibrillin-1 microfibril formation in connective tissues. Results: Successful improvement of Marfan syndrome manifestations are induced by the direct administration of recombinant ADAMTSL6β. Conclusion: This study demonstrated critical importance of microfibril regeneration in preventing Marfan syndrome. Significance: Our current data support a new concept that the regeneration of microfibrils using ADAMTSL6β is essential for improving Marfan syndrome. Marfan syndrome (MFS) is a systemic disorder of the connective tissues caused by insufficient fibrillin-1 microfibril formation and can cause cardiac complications, emphysema, ocular lens dislocation, and severe periodontal disease. ADAMTSL6β (A disintegrin-like metalloprotease domain with thrombospondin type I motifs-like 6β) is a microfibril-associated extracellular matrix protein expressed in various connective tissues that has been implicated in fibrillin-1 microfibril assembly. We here report that ADAMTSL6β plays an essential role in the development and regeneration of connective tissues. ADAMTSL6β expression rescues microfibril disorder after periodontal ligament injury in an MFS mouse model through the promotion of fibrillin-1 microfibril assembly. In addition, improved fibrillin-1 assembly in MFS mice following the administration of ADAMTSL6β attenuates the overactivation of TGF-β signals associated with the increased release of active TGF-β from disrupted fibrillin-1 microfibrils within periodontal ligaments. Our current data thus demonstrate the essential contribution of ADAMTSL6β to fibrillin-1 microfibril formation. These findings also suggest a new therapeutic strategy for the treatment of MFS through ADAMTSL6β-mediated fibrillin-1 microfibril assembly.

Differential responses to parathyroid hormone-related protein (PTHrP) deficiency in the various craniofacial cartilages

PTHrP null mutant mice exhibit skeletal abnormalities both in the craniofacial region and limbs. In the growth plate cartilage of the null mutant, a diminished number of proliferating chondrocytes and accelerated chondrocytic differentiation are observed. In order to examine the effect of PTHrP deficiency on the craniofacial morphology and highlight the differential feature of the composing cartilages, we examined the various cartilages in the craniofacial region of neonatal PTHrP deficient mice. The major part of the cartilaginous anterior cranial base appeared to be normal in the homozygous PTHrP deficient mice. However, acceleration of chondrocytic differentiation and endochondral bone formation was observed in the posterior part of the anterior cranial base and in the cranial base synchondroses. Ectopic bone formation was observed in the soft tissue‐running mid‐portion of the Meckels cartilage, where the cartilage degenerates and converts to ligament in the course of normal development. The zonal structure of the mandibular condylar cartilage was scarcely affected, but the whole condyle was reduced in size. These results suggest the effect of PTHrP deficiency varies widely between the craniofacial cartilages, according to the differential features of each cartilage. Anat Rec 255:452–457, 1999.

Non-syndromic Oligodontia with a Novel Mutation of PAX9

Agenesis of the permanent teeth is a congenital anomaly that is frequently seen in humans. Oligodontia is a severe type of tooth agenesis involving 6 or more congenitally missing teeth, excluding the third molars. Previous studies have indicated that mutations in the homeobox gene MSX1, paired domain transcription factor PAX9, and EDA are associated with non-syndromic oligodontia. This study reports a Japanese family (eight of 14 family members affected) with non-syndromic oligodontia who preferentially lacked molar teeth. In this family, a novel frameshift mutation (321_322insG) was identified in the paired domain of PAX9. The frameshift mutation caused altered amino acids in the paired domain and premature termination of translation by 26 amino acids. When transfected into COS-7 cells, the mRNA expression of 321_322insG PAX9 was comparable with that of wild-type PAX9. However, the mRNA of 321_322insG PAX9 was more unstable than that of wild-type PAX9. This mRNA instability caused a marked decrease in protein production, as evaluated by Western blot analysis and immunostaining. These findings suggest that the 321_322insG mutation causes insufficient function of PAX9 protein and haploinsufficiency as a genetic model of familial non-syndromic oligodontia with a PAX9 mutation.