Marine collagen derived from fish scales, skin, and bone has been widely investigated for application as a scaffold and carrier due to its bioactive properties, including excellent biocompatibility, low antigenicity, and high biodegradability and cell growth potential. Fish type I collagen is an effective material as a biodegradable scaffold or spacer replicating the natural extracellular matrix, which serves to spatially organize cells, providing them with environmental signals and directing site-specific cellular regulation. This study was conducted to confirm the safety of fish (tilapia) atelocollagen for use in clinical application. We performed in vitro and in vivo biological studies of medical materials to investigate the safety of fish collagen. The extract of fish collagen gel was examined to clarify its sterility. All present sterility tests concerning bacteria and viruses (including endotoxin) yielded negative results, and all evaluations of cell toxicity, sensitization, chromosomal aberrations, intracutaneous reactions, acute systemic toxicity, pyrogenic reactions, and hemolysis were negative according to the criteria of the ISO and the Ministry of Health, Labour and Welfare of Japan. The present study demonstrated that atelocollagen prepared from tilapia is a promising biomaterial for use as a scaffold in regenerative medicine.

The strict class system in place during the Edo (early modern) period in Japan is thought to have encompassed customs that differed between the samurai (ruling class) and commoners. This study found that in samurai children, deciduous caries occurred only in maxillary incisors at 0.5-2 years of age and did not occur in the mandibular incisors and canines at 3-5 years of age. Conversely, in commoner children, deciduous caries occurred in all maxillary teeth and mandibular molars at 0.5-2 years of age and in all maxillary and mandibular teeth at 3-5 years of age. In commoner children, deciduous caries was seen in tooth types that have a low incidence of deciduous caries in modern Japanese. The present results show that deciduous carious lesions appear earlier and more frequently in the commoner children compared to the samurai children, probably because of differences in lifestyle between the two groups. There was no significant difference in the incidence of enamel hypoplasia in the deciduous dentition between samurai and commoner children, suggesting that the two classes had broadly comparable nutrition. In our previous study using the same skeletal series, similar results between samurai and commoners were found with respect to caries and enamel hypoplasia prevalence of the permanent dentition. Many of the samurai adults had slick polished teeth, which we believe were caused by regular brushing of the teeth in the samurai class. Such a custom might have functioned to prevent dental caries not only in the permanent dentition, but also in the deciduous dentition. Thus, oral hygiene practices in the samurai class are thought to have functioned to prevent dental caries not only in adults but also in children.

A low concentration of boron (B) accelerates the proliferation and differentiation of mammalian osteoblasts. The aim of this study was to investigate the effects of 0.1 mM of B on the membrane function of osteoblastic cells in vitro. Genes involved in cell activity were investigated using gene expression microarray analyses. The Ca influx and efflux were evaluated to demonstrate the activation of L-type Ca channel for the Ca influx, and that of Na/K-ATPase for the Ca efflux. A real-time PCR analysis revealed that the messenger RNA (mRNA) expression of four mineralization-related genes was clearly increased after 3 days of culture with a B-supplemented culture medium. Using microarray analyses, five genes involved in cell proliferation and differentiation were upregulated compared to the control group. Regarding the Ca influx, in the nifedipine-pretreated group, the relative fluorescence intensity for 1 min after adding B solution did not increase compared with that for 1 min before addition. In the control group, the relative fluorescence intensity was significantly increased compared with the experimental group (P < 0.05). Regarding the Ca efflux, in the experimental group cultured in 0.1 mM of B-supplemented medium, the relative fluorescence intensity for 10 min after ouabain treatment revealed a significantly lower slope value compared with the control group (P < 0.01). This is the first study to demonstrate the acceleration of Ca flux by B supplementation in osteoblastic cells. Cell membrane stability is related to the mechanism by which a very low concentration of B promotes the proliferation and differentiation of mammalian osteoblastic cells in vitro.

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Retroviral transduction of four transcription factors (Oct4, Sox2, Klf4 and c-Myc) or three factors, excluding c-Myc, has been shown to initiate a reprogramming process that results in the transformation of murine fibroblasts to induced pluripotent stem (iPS) cells, and there has been a rapid increase in the number of iPS cell-based preclinical trials. In this study, the effects of these transcription factors were evaluated regarding the growth and differentiation of murine iPS cells under hypoxia. Based on the results of RT-PCR and alizarin red S staining, there were no statistical differences in the growth and differentiation of iPS cells or the induction of iPS cells to osteoblasts under hypoxia between the transcription factor groups. Furthermore, the function of hypoxia inducible factors (HIFs) in murine iPS cells under hypoxia was investigated in relation to the morphology and expression of transcription factors using RT-PCR and Western blotting. The HIF-2α knockdown group exhibited a decrease in the colony size of the iPS cells. The HIF-2α or -3α knockdown group demonstrated a statistically significant decrease in the transcription factor expression compared to that observed in the control group. These results demonstrate that HIF-2α among HIFs is the most influential candidate for the maintenance of the pluripotency of murine iPS cells.

A hypoxic condition is known to contribute to pluripotency. In the present article, the effects of transcription factors were first assessed regarding the proliferation and differentiation of human induced pluripotent stem (iPS) cells under hypoxic conditions using cell morphology and real-time polymerase chain reaction (RT-PCR). Morphology evaluations and RT-PCR revealed that the colony formation was promoted and the expression of pluripotent markers was increased under hypoxic conditions. In addition, the function of hypoxia inducible factors (HIFs) in human iPS cells under hypoxic conditions was evaluated in relation to the morphology and the expression of pluripotency markers by siRNA and RT-PCR. The HIF-2α silencing group showed a reduction in the colony size of human iPS cells and a statistically significant reduction in the expression of undifferentiation markers compared to the control group. Furthermore, the expression of HIF-2α was decreased when signal transducer and activator of transcription 3 (STAT3) was suppressed by its inhibitor, Stattic or S31 201. The inhibition using Stattic did not produce colony formation. The expression of pluripotent markers was also decreased using Stattic or S31 201. This study indicates that the HIF-2α expression in human iPS cells was activated under hypoxic conditions, similarly to that in murine iPS cells, and that HIF-2α among HIFs is the most effective compound for maintaining the pluripotency of human iPS cells. Furthermore, the STAT3 signal pathway regulates the expression of HIF-2α.

The family register system used by the Tokugawa shogunate limited the ability of commoners to move freely from one location to another. It is thought that such restrictions on movement resulted in regional differences in lifestyle, including diet, arising from regional environmental differences. It is also likely that regional differences in lifestyle resulted in regional differences in health status, including oral health. In the present study, we examined differences in the dental pathology of commoners from two early modern settlements in Japan-the Kyoumachi site in Kokura, Fukuoka Prefecture and the Miwanoyama site in Nagareyama, Chiba Prefecture-as a preliminary analysis of regional differences in the dental pathology of early modern Japanese. We found that the prevalence of root caries and antemortem tooth loss (AMTL) were significantly higher in Miwanoyama commoners than in Kyoumachi commoners. In addition, the prevalence of severe enamel hypoplasia (EHP) in Miwanoyama commoners was significantly higher than that in Kyoumachi commoners, and the dental attrition of the Miwanoyama commoners was more severe than that of the Kyoumachi commoners. The present results therefore reveal considerable differences in dental pathology between commoners who lived in different areas. The high prevalence of root caries and AMTL in Miwanoyama suggests that periodontal disease spread among the commoners at this site, and the high prevalence of severe EHP and dental attrition in Miwanoyama suggests that the dietary status of the Miwanoyama commoners was inferior to that of the Kyoumachi commoners. We conclude that pathological differences in oral health status between the Miwanoyama and Kyoumachi commoners was most likely influenced by differences in lifestyle, including diet, arising from regional environmental differences.

We analyzed the mitochondrial DNA extracted from 14 human skeletal remains from the Doigahama site in Japan to clarify the genetic structure of the Doigahama Yayoi population and the relationship between burial style and kinship among individuals. The sequence types obtained in this study were compared with those of the modern Japanese, northern Kyushu Yayoi and ancient Chinese populations. We found that the northern Kyushu Yayoi populations belonged to the groups that include most of the modern Japanese population. In contrast, most of the Doigahama Yayoi population belonged to the group that includes a small number of the modern Japanese population. These results suggest that the Doigahama Yayoi population might have contributed less to the formation of the modern Japanese population than the northern Kyushu Yayoi populations. Moreover, when we examined the kinship between individuals in the Doigahama site, we found that the vicinal burial of adult skeletons indicated a maternal kinship, although that of juvenile skeletons did not. The vicinal burial style might have been influenced by many factors, such as paternal lineages, periods and geographical regions, as well as maternal lineages. In addition, skeletons considered to be those of shamans or leaders had the same sequence types. Their crucial social roles may have been inherited through maternal lineage.