Articular cartilage and synovial joints are critical for skeletal function, but the mechanisms regulating their development are largely unknown. In previous studies we found that the ets transcription factor ERG and its alternatively-spliced variant C-1-1 have roles in joint formation in chick. Here, we extended our studies to mouse. We found that ERG is also expressed in developing mouse limb joints. To test regulation of ERG expression, beads coated with the joint master regulator protein GDF-5 were implanted close to incipient joints in mouse limb explants; this led to rapid and strong ectopic ERG expression. We cloned and characterized several mammalian ERG variants and expressed a human C-1-1 counterpart (hERG3Delta81) throughout the cartilaginous skeleton of transgenic mice, using Col2a1 gene promoter/enhancer sequences. The skeletal phenotype was severe and neonatal lethal, and the transgenic mice were smaller than wild type littermates and their skeletons were largely cartilaginous. Limb long bone anlagen were entirely composed of chondrocytes actively expressing collagen IX and aggrecan as well as articular markers such as tenascin-C. Typical growth plates were absent and there was very low expression of maturation and hypertrophy markers, including Indian hedgehog, collagen X and MMP-13. The results suggest that ERG is part of molecular mechanisms leading chondrocytes into a permanent developmental path and become joint forming cells, and may do so by acting downstream of GDF-5.

The retinoic acid receptors α, β and γ (RARα, RARβ and RARγ) are nuclear hormone receptors that regulate fundamental processes during embryogenesis, but their roles in skeletal development and growth remain unclear. To study skeletal-specific RAR function, we created conditional mouse mutants deficient in RAR expression in cartilage. We find that mice deficient in RARα and RARγ (or RARβ and RARγ) exhibit severe growth retardation obvious by about 3 weeks postnatally. Their growth plates are defective and, importantly, display a major drop in aggrecan expression and content. Mice deficient in RARα and RARβ, however, are virtually normal, suggesting that RARγ is essential. In good correlation, we find that RARγ is the most strongly expressed RAR in mouse growth plate and its expression characterizes the proliferative and pre-hypertrophic zones where aggrecan is strongly expressed also. By being avascular, those zones lack endogenous retinoids and thus RARγ is likely to exert ligand-less repressor function. Indeed, our data indicate that: aggrecan production is enhanced by RARγ over-expression in chondrocytes under retinoid-free culture conditions; production is further boosted by corepressor Zac1 or pharmacologic agents that enhance RAR repressor function; and RAR/Zac1 function on aggrecan expression may involve Sox proteins. In sum, our data reveal that RARs, and RARγ in particular, exert previously unappreciated roles in growth plate function and skeletal growth and regulate aggrecan expression and content. Since aggrecan is critical for growth plate function, its deficiency in RAR-mutant mice is likely to have contributed directly to their growth retardation.

In this paper we investigate the evolutionary changes of chess variants. For this purpose we propose a measure of the game's entertaining impact. The measure is derived from grandmaster games and is applicable to chess variants independent of the game under consideration. For the case where no grandmaster games are available we perform computer self-play experiments to estimate the measure. For several old chess variants the measure as well as other measures, such as the search-space complexity and draw rate, are calculated. Some relationships between the measures investigated are found. Based on these relationships the evolutionary changes of chess variants are discussed.Keywords: entertainment measure, evolution of chess variants Introd uctionIn computer-games related studies the complexity of the game is an important issue. We can distinguish at least two types of complexity: search-space complexity and decision complexity. The decision complexity depends on the intricacies of a given game, such as the rules, the possible strategies and the depth of search. The measure of decision complexity is highly subjective and differs from game to game, even if they use the same class within chess variants. For an adequate comparison of the measures of complexity of several chess variants, we restrict ourselves to a measure of the search-space complexity [l]. In a first approximation this complexity can be measured as the size of a minimax search tree necessary for solving the game. The complexity measure is then given by the value BD in which B is the average number of possible moves and D is the average game length.

Several recent prospective clinical trials have investigated the effect of supplementary vibration applied with fixed appliances in an attempt to accelerate tooth movement and shorten the duration of orthodontic treatment. Among them, some studies reported an increase in the rate of tooth movement, but others did not. This technique is still controversial, and the underlying cellular and molecular mechanisms remain unclear. In the present study, we developed a new vibration device for a tooth movement model in rats, and investigated the efficacy and safety of the device when used with fixed appliances. The most effective level of supplementary vibration to accelerate tooth movement stimulated by a continuous static force was 3 gf at 70 Hz for 3 minutes once a week. Furthermore, at this optimum-magnitude, high-frequency vibration could synergistically enhance osteoclastogenesis and osteoclast function via NF-κB activation, leading to alveolar bone resorption and finally, accelerated tooth movement, but only when a static force was continuously applied to the teeth. These findings contribute to a better understanding of the mechanism by which optimum-magnitude high-frequency vibration accelerates tooth movement, and may lead to novel approaches for the safe and effective treatment of malocclusion.

The ability of cells to sense and respond to physical stress is required for tissue homeostasis and normal development. In muscle, bone, tendon, periodontium, and the cardiovascular system, applied forces of physiological magnitude regulate cellular processes that are critical for normal tissue and organ functions, such as differentiation, proliferation, and migration (1). The periodontal ligament (PDL) 3 is a connective tissue interposed between the roots of teeth and the inner wall of the tooth-supporting bone (alveolar bone) socket. The PDL constitutively and iatrogenically receives mechanical stress, such as occlusal pressure and orthodontic forces, which have effects on the homeostasis of the PDL (2). Proper mechanical stress on teeth induces not only the proliferation and differentiation of PDL cells into osteoblasts and cementoblasts but also the synthesis and degradation of extracellular matrix (ECM) molecules (3). For example, during orthodontic tooth movement, two types of sites (tension sites and pressure sites) arise around the tooth through the orthodontic force. At the tension sites, the PDL is stretched, and the expressions of bone-related genes, such as osteocalcin (4) and bone sialoprotein (5), are up-regulated, such that bone formation is finally induced on the alveolar bone facing the tooth root (6). On the other hand, at the pressure sites, the PDL is compressed, and osteoclasts are activated. Consequently, resorption of the alveolar bone is induced. An orchestrated balance between bone formation and resorption controls tooth movement (7). In contrast, elimination of mechanical stress on teeth is known to cause atrophy of the PDL in vivo (8). Kaneko et al. (9) reported that loss of occlusal function by extraction of the antagonistic upper molars of rats caused atrophic changes in the PDL of the lower molars, such as narrowing of the space, disorientation of collagen fibers, and decreases in proteoglycans. These findings indicate that mechanical stress on teeth affects the remodeling of the PDL, cementum, and alveolar bone. Thus, it is important to clarify the physiological functions of mechanical stress on the PDL.To clarify the molecular basis of the mechanical stress-regulated PDL functions, we analyzed the gene expression profile of human PDL cells receiving tensile mechanical stress in vitro. Interestingly, an oligo-DNA chip analysis identified two glutamate signaling-associated genes, HOMER1 (homer homolog 1) and GRIN3A (glutamate receptor ionotropic N-methyl-D-aspartate 3A), among the up-regulated genes. L-Glutamate is the most abundant amino acid in the central nervous system and plays important roles in neurotransmission (10

Osteocytes produce various factors that mediate the onset of bone formation and resorption and play roles in maintaining bone homeostasis and remodeling in response to mechanical stimuli. One such factor, CCN2, is thought to play a significant role in osteocyte responses to mechanical stimuli, but its function in osteocytes is not well understood. Here, we showed that CCN2 induces apoptosis in osteocytes under compressive force loading. Compressive force increased CCN2 gene expression and production, and induced apoptosis in osteocytes. Application of exogenous CCN2 protein induced apoptosis, and a neutralizing CCN2 antibody blocked loading-induced apoptosis. We further examined how CCN2 induces loaded osteocyte apoptosis. In loaded osteocytes, extracellular signal-regulated kinase 1/2 (ERK1/2) was activated, and an ERK1/2 inhibitor blocked loading-induced apoptosis. Furthermore, application of exogenous CCN2 protein caused ERK1/2 activation, and the neutralizing CCN2 antibody inhibited loading-induced ERK1/2 activation. Therefore, this study demonstrated for the first time to our knowledge that enhanced production of CCN2 in osteocytes under compressive force loading induces apoptosis through activation of ERK1/2 pathway.

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The field of tooth regeneration has progressed in recent years, and human tooth regeneration could become viable in the future. Because induced pluripotent stem (iPS) cells can differentiate into odontogenic cells given appropriate conditions, iPS cells are a potential cell source for tooth regeneration. However, a definitive method to induce iPS cell-derived odontogenic cells has not been established. We describe a novel method of odontoblast differentiation from iPS cells using gene transfection. We generated mouse iPS cell-derived neural crest-like cells (iNCLCs), which exhibited neural crest markers. Next, we differentiated iNCLCs into odontoblast-like cells by transfection of Pax9 and Bmp4 expression plasmids. Exogenous Pax9 upregulated expression of Msx1 and dentin matrix protein 1 (Dmp1) in iNCLCs but not bone morphogenetic protein 4 (Bmp4) or dentin sialophosphoprotein (Dspp). Exogenous Bmp4 upregulated expression of Msx1, Dmp1, and Dspp in iNCLCs, but not Pax9. Moreover, cotransfection of Pax9 and Bmp4 plasmids in iNCLCs revealed a higher expression of Pax9 than when Pax9 plasmid was used alone. In contrast, exogenous Pax9 downregulated Bmp4 overexpression. Cotransfection of Pax9 and Bmp4 synergistically upregulated Dmp1 expression; however, Pax9 overexpression downregulated exogenous Bmp4-induced Dspp expression. Together, these findings suggest that an interaction between exogenous Pax9-and Bmp4-induced signaling modulated Dmp1 and Dspp expression. In conclusion, transfection of Pax9 and Bmp4 expression plasmids in iNCLCs induced gene expression associated with odontoblast differentiation, suggesting that iNCLCs differentiated into odontoblast-like cells. The iPS cellderived odontoblast-like cells could be a useful cell source for tooth regeneration. STEM CELLS TRANSLATIONAL MEDICINE 2015;4:993-997