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The differentiation of mesenchymal cells into chondrocytes and chondrocyte proliferation and maturation are fundamental steps in skeletal development. Runx2 is essential for osteoblast differentiation and is involved in chondrocyte maturation. Although chondrocyte maturation is delayed inVertebrate skeletons are constructed through the formation of bone structures, a process that is achieved by intramembranous or endochondral ossification. Intramembranous bones, which are directly formed by osteoblasts, are restricted to the cranial vault, some facial bones, and parts of the mandible and clavicle, whereas the rest of the skeleton is composed of endochondral bones that are formed as a cartilaginous template which is then replaced by bone. In early skeletal development, mesenchymal cells condense and acquire the phenotypes of chondrocytes including the ability to produce Col2a1 and proteoglycan. In the process of endochondral ossification, immature chondrocytes proliferate, and chondrocytes at the center of the cartilaginous skeleton begin to mature to become prehypertrophic chondrocytes, which express parathyroid hormone/parathyroid hormone-related peptide (Pthlh) receptor (Pthr1) and Indian hedgehog (Ihh). The prehypertrophic chondrocytes further mature to hypertrophic chondrocytes, which express Col10a1. Upon the terminal differentiation of chondrocytes, the terminal hypertrophic chondrocytes express osteopontin, the matrix is mineralized, vascular vessels invade the calcified cartilage, and finally the cartilage is replaced by bone. Chondrocyte proliferation and differentiation occur in an organized manner and result in the formation of a growth plate that is composed of layers of chondrocytes at different stages of differentiation, in-

During skeletogenesis, cartilage develops to either permanent cartilage that persists through life or transient cartilage that is eventually replaced by bone. However, the mechanism by which cartilage phenotype is specified remains unclarified. Core binding factor α1 (Cbfa1) is an essential transcription factor for osteoblast differentiation and bone formation and has the ability to stimulate chondrocyte maturation in vitro. To understand the roles of Cbfa1 in chondrocytes during skeletal development, we generated transgenic mice that overexpress Cbfa1 or a dominant negative (DN)-Cbfa1 in chondrocytes under the control of a type II collagen promoter/enhancer. Both types of transgenic mice displayed dwarfism and skeletal malformations, which, however, resulted from opposite cellular phenotypes. Cbfa1 overexpression caused acceleration of endochondral ossification due to precocious chondrocyte maturation, whereas overexpression of DN-Cbfa1 suppressed maturation and delayed endochondral ossification. In addition, Cbfa1 transgenic mice failed to form most of their joints and permanent cartilage entered the endochondral pathway, whereas most chondrocytes in DN-Cbfa1 transgenic mice retained a marker for permanent cartilage. These data show that temporally and spatially regulated expression of Cbfa1 in chondrocytes is required for skeletogenesis, including formation of joints, permanent cartilages, and endochondral bones.

Core-binding factor beta (CBFbeta, also called polyomavirus enhancer binding protein 2beta (PEBP2B)) is associated with an inversion of chromosome 16 and is associated with acute myeloid leukemia in humans. CBFbeta forms a heterodimer with RUNX1 (runt-related transcription factor 1), which has a DNA binding domain homologous to the pair-rule protein runt in Drosophila melanogaster. Both RUNX1 and CBFbeta are essential for hematopoiesis. Haploinsufficiency of another runt-related protein, RUNX2 (also called CBFA1), causes cleidocranial dysplasia in humans and is essential in skeletal development by regulating osteoblast differentiation and chondrocyte maturation. Mice deficient in Cbfb (Cbfb(-/-)) die at midgestation, so the function of Cbfbeta in skeletal development has yet to be ascertained. To investigate this issue, we rescued hematopoiesis of Cbfb(-/-) mice by introducing Cbfb using the Gata1 promoter. The rescued Cbfb(-/-) mice recapitulated fetal liver hematopoiesis in erythroid and megakaryocytic lineages and survived until birth, but showed severely delayed bone formation. Although mesenchymal cells differentiated into immature osteoblasts, intramembranous bones were poorly formed. The maturation of chondrocytes into hypertrophic cells was markedly delayed, and no endochondral bones were formed. Electrophoretic mobility shift assays and reporter assays showed that Cbfbeta was necessary for the efficient DNA binding of Runx2 and for Runx2-dependent transcriptional activation. These findings indicate that Cbfbeta is required for the function of Runx2 in skeletal development.

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The fundamental issues on the smallest possible processing accuracy and the best feasible surface smoothness in pinpoint polymerization-based laser fabrication were experimentally investigated. The lateral spatial resolution is improved from the previously reported value, 120nm, to around 100nm by intentionally introducing radical quenchers in the resin. The roughness measured from 10μm×10μm surface areas were averaged to 4–11nm, which is found slightly affected by the laser pulse energy but independent on the scanning pitch when it is smaller than a critical value. The surface quality of this level could fully satisfy the requirement of various photonic elements and devices.

Objective: To investigate the laterality of the normal asymmetry of the human face, examining differences in laterality in relation to sex, growth stage, and skeletal classification. Materials and Methods: A total of 1800 Japanese subjects (651 males and 1149 females; mean age, 15 years 3 months; range, 4 years 2 months to 59 years 11 months) were selected. Individuals in the sample were categorized according to sex, one of three growth stages, and one of three skeletal patterns. Differences in length between distances from the points at which ear rods were inserted to the facial midline and the perpendicular distance from the soft-tissue menton to the facial midline were measured on a frontal facial photograph. Subjects with a discrepancy of more than 3 standard deviations of the measurement error were categorized as having left-or right-sided laterality. Results: Of subjects with facial asymmetry, 79.7% had a wider right hemiface, and 79.3% of those with chin deviation had left-sided laterality. These tendencies were independent of sex, age, or skeletal jaw relationships. In this regard, during pubertal growth, the proportion of subjects with wider right hemiface decreased (P Ͻ .0001), whereas the proportion of those with a wider left hemiface increased (P Ͻ .01), despite a consistent tendency for right-sided dominance. Conclusion: These results suggest that laterality in the normal asymmetry of the face, which is consistently found in humans, is likely to be a hereditary rather than an acquired trait.

Receptor activator of nuclear factor-B ligand (RANKL), osteoprotegerin (OPG), and macrophage-colony stimulating factor play essential roles in the regulation of osteoclastogenesis. Runx2-deficient (Runx2 ؊/؊ ) mice showed a complete lack of bone formation because of maturational arrest of osteoblasts and disturbed chondrocyte maturation. Further, osteoclasts were absent in these mice, in which OPG and macrophage-colony stimulating factor were normally expressed, but RANKL expression was severely diminished. We investigated the function of Runx2 in osteoclast differentiation. A Runx2 ؊/؊ calvaria-derived cell line (CA120 -4), which expressed OPG strongly but RANKL barely, severely suppressed osteoclast differentiation from normal bone marrow cells in co-cultures. Adenoviral introduction of Runx2 into CA120 -4 cells induced RANKL expression, suppressed OPG expression, and restored osteoclast differentiation from normal bone marrow cells, whereas the addition of OPG abolished the osteoclast differentiation induced by Runx2. Addition of soluble RANKL (sRANKL) also restored osteoclast differentiation in co-cultures. Forced expression of sRANKL in Runx2 ؊/؊ livers increased the number and size of osteoclast-like cells around calcified cartilage, although vascular invasion into the cartilage was superficial because of incomplete osteoclast differentiation. These findings indicate that Runx2 promotes osteoclast differentiation by inducing RANKL and inhibiting OPG. As the introduction of sRANKL was insufficient for osteoclast differentiation in Runx2 ؊/؊ mice, however, our findings also suggest that additional factor(s) or matrix protein(s), which are induced in terminally differentiated chondrocytes or osteoblasts by Runx2, are required for osteoclastogenesis in early skeletal development.In the process of endochondral ossification, chondrocytes mature to hypertrophic chondrocytes, matrix around terminally differentiated chondrocytes (terminal hypertrophic chondrocytes) is mineralized, blood vessels invade into the calcified cartilage, and cartilage is replaced by bone (1). Osteoclasts accelerate these processes by resorption of the calcified matrix leading to bone marrow formation. Osteoclasts differentiate from hematopoietic precursor cells through direct contact with osteoblastic/stromal cells (2). Recently, osteoprotegerin (OPG) 1 / osteoclastogenesis inhibitory factor, which is an inhibitor of osteoclast differentiation (3, 4), and receptor activator of NF-B (RANK) ligand (RANKL)/tumor necrosis factor-related activation-induced cytokine/OPG ligand/osteoclast differentiation factor, which is an inducer of osteoclast differentiation (5-8), were identified. RANKL, which is expressed on the surface of osteoblastic/stromal cells or released as a soluble factor, binds to its receptor RANK (9, 10), which is expressed on the surface of osteoclast precursors and osteoclasts, and induces osteoclast differentiation and activation. OPG, which binds RANKL with higher affinity than RANK, acts as a decoy receptor for RANKL and in...