The Wnt/ß-catenin signaling pathway has a healing ability for periapical periodontitis.

Various disease-related genes have recently been identified using single nucleotide polymorphisms (SNPs). This study identified disease-related genes by analyzing SNP using genomic DNA isolated from Japanese patients with periapical periodontitis. Results showed that the SNP in LRP5 demonstrated a significant genotypic association with periapical lesions (Fisher's exact test, P < 0.05). We constructed an in vivo murine periapical periodontitis model to confirm the Wnt/ß-catenin signaling pathway's role in developing and healing periapical periodontitis. We observed that administration of the Wnt/ß-catenin signaling pathway inhibitor enlarged the periapical lesion. Moreover, applying lithium chloride (LiCl) to root canals accelerated periapical periodontitis healing. Histological analysis demonstrated that the expression levels of Col1a1 and Runx2 increased in the LiCl application group compared to that in the control group. Furthermore, many CD45R-positive cells appeared in the periapical lesions in the LiCl application group. These results indicated that LiCl promoted the healing of periapical periodontitis by inducing bone formation and immune responses. Our findings suggest that the Wnt/ß-catenin signaling pathway regulates the development of periapical periodontitis. We propose a bioactive next-generation root canal treatment agent for this dental lesion.

Intracellular density is a novel indicator of differentiation stages of murine osteoblast lineage cells.

Osteoblasts are primary bone-making cells originating from mesenchymal stem cells (MSCs) in the bone marrow. The differentiation of MSCs to mature osteoblasts involves an intermediate stage called preosteoblasts, but the details of this process remain unclear. This study focused on the intracellular density of immature osteoblast lineage cells and hypothesized that the density might vary during differentiation and might be associated with the differentiation stages of osteoblast lineage cells. This study aimed to clarify the relationship between intracellular density and differentiation stages using density gradient centrifugation. Primary murine bone marrow stromal cell cultures were prepared in an osteogenic induction medium, and cells were separated into three fractions (low, intermediate, and high-density). The high-density fraction showed elevated expression of osteoblast differentiation markers (Sp7, Col1a1, Spp1, and Bglap) and low expression of MSC surface markers (Sca-1, CD73, CD105, and CD106). In contrast, the low-density fraction showed a high expression of MSC surface markers. These results indicated that intracellular density increased during differentiation from preosteoblasts to committed osteoblasts. Intracellular density may be a novel indicator for osteoblast differentiation stages. Density gradient centrifugation is a novel technique to study the process by which preosteoblasts transform into bone-forming cells.

Kruppel-Like Factor 4 represses osteoblast differentiation via ciliary Hedgehog signaling.

Primary cilia are appendages observed in most types of cells, and serve as cellular antennae for sensing environmental signals. Evidence is accumulating that correct ciliogenesis and ciliary functions are indispensable for normal skeletal development by regulating signaling pathways important for bone development. However, whether ciliogenesis is regulated by bone-related factors in osteoblasts is largely unknown. Here we show that Kruppel-Like Factor 4 (KLF4), which is known to repress osteoblast differentiation, supports the formation and maintenance of cilia in cultured osteoblasts; however, the length of the cilia observed in KLF4-induced cells were significantly shorter compared to the control cells. Basal Hedgehog signaling was repressed by KLF4. Significantly, activating Hedgehog signaling using a Smoothened agonist significantly rescued osteoblast mineralization and osteoblastic gene expressions. Global gene expression analysis showed that KLF4 induced number of genes including the nuclear receptor, Pregnane X receptor (PXR), and PXR repressed calvarial osteoblast mineralization and repressed Gli1 expression similar as the effect observed by inducing KLF4. Our results implicate that KLF4 plays important roles for maintaining osteoblasts in an immature state by repressing basal activation of the Hedgehog signaling.

Factors that cause endodontic failures in general practices in Japan.

BACKGROUND: Bacterial biofilms that develop on root surfaces outside apical foramens have been found to be associated with refractory periapical periodontitis. However, several other factors cause endodontic failures apart from extraradicular biofilms. The aim of this study was to identify the factors causing endodontic failures in general practices in Japan. METHODS: Patients diagnosed as having refractory periapical periodontitis by general practitioners and who requested endodontic treatment at Osaka University Dental Hospital were selected by checking medical records from April 2009 to March 2013. Factors causing endodontic failures were identified. RESULTS: A total of 103 teeth were selected, and 76 teeth completed root-canal treatment. Tooth extractions were required for 18 teeth after or without endodontic treatment. Six teeth required apicoectomy after endodontic treatment. One tooth needed hemisection. One tooth needed intentional replantation. One tooth needed adhesion and replantation. The main causes of treatment failure were open apices (24 teeth), perforation (18 teeth), and root fracture (13 teeth). In six teeth with open apices that required apicoectomy or extraction, extraradicular biofilms may have been related to endodontic failure. CONCLUSIONS: Most endodontic cases diagnosed with refractory periapical periodontitis by general practitioners were compromised by any other factors rather than extraradicular biofilms.

HipOP mesenchymal population has high potential for repairing injured peripheral nerves.

Bone marrow stromal cells (BMSCs) are reportedly a heterogeneous population of mesenchymal stem cells (MSCs). Recently, we developed a simple strategy for the enrichment of MSCs with the capacity to differentiate into osteoblasts, chondrocytes, and adipocytes. On transplantation, the progenitor-enriched fractions can regenerate the bone with multiple lineages of donor origin and are thus called "highly purified osteoprogenitors" (HipOPs). Although our previous studies have demonstrated that HipOPs are enriched with MSCs and exhibit a higher potential to differentiate into osteoblasts, adipocytes, and chondrocytes than BMSCs, their potential to differentiate into neural cells has not been clarified. In this study, we evaluated the efficacy of HipOPs as a resource of neural stem cells. The neurosphere assay showed that neurospheres formed by HipOPs exhibited self-renewal ability and their size was generally larger than that of neurospheres formed by BMSCs. A limiting dilution assay was used to evaluate the frequency of neural progenitors in BMSCs and HipOPs. The results demonstrated that the frequency of neural progenitors in HipOPs was 120-fold higher than that in BMSCs. Furthermore, to investigate the in vivo regenerative potential of the peripheral nerve, we modified a murine peripheral nerve injury experimental model and demonstrated that HipOPs exhibit a higher efficacy in repairing injured peripheral nerves. These findings suggest that HipOPs are a useful cell resource for regenerative therapies such as that in case of peripheral nerve injury.

Developmental Stage-Dependent Effects of Leukemia Inhibitory Factor on Adipocyte Differentiation of Murine Bone Marrow Stromal Cells.

Studies describing the effects of leukemia inhibitory factor (LIF) on adipocyte differentiation in murine cells have shown varying results. For example, LIF has been reported to have a suppressive effect on adipocyte differentiation in the 3T3-L1 cell line, whereas it promoted adipocyte differentiation in the Ob1771 and 3T3-F442A cell lines. Thus, it is possible that the effects of LIF on adipogenesis vary with the developmental stage of the cells or tissues, but the details remain unclear. To further elucidate the role of LIF in adipogenesis, we investigated the effects of LIF on murine bone marrow stromal cells at the early and late stages of adipogenesis. LIF decreased the number of lipid foci and suppressed the expression levels of adipocyte differentiation markers at day 5; however, it enhanced these same traits at day 15. A previous report showed that the expression levels of Wnt signaling molecules are different at the early and late differentiation stages; therefore, we investigated the relationship between LIF and Wnt signaling. LIF affected the mRNA expression levels of different Wnt signaling molecules but inhibited the expression level of ß-catenin protein at both days 5 and 15. Our data suggest that LIF has reciprocal roles during the early and late stages of adipocyte differentiation, regulating the Wnt signaling pathway.

Density Gradient Centrifugation for the Isolation of Cells of Multiple Lineages.

We recently developed a simple strategy for the enrichment of mesenchymal stem cells (MSCs) with the capacity for osteoblast, chondrocyte, and adipocyte differentiation. On transplantation, the progenitor-enriched fraction can regenerate bone with multiple lineages of donor origin. Although comprising multiple precursor cell types, the population is enriched >100-fold in osteoprogenitors, hence the name "highly purified osteoprogenitors" (HipOPs). To establish a new modified method of purifying pure MSCs, it is useful to know the expression patterns of surface markers on heterogeneous MSCs and committed cells such as osteoblasts, adipocytes, and chondrocytes. However, calcium deposition by osteoblasts is a critical obstacle in visualizing the expression patterns of surface markers. We now report a new method of separating differentiated osteoblastic HipOPs (OB-HipOPs) from calcium deposits using the Percoll density gradient centrifugation technique. After centrifuge separation, calcium deposits were observed at the bottom of the centrifuge tube, and living OB-HipOPs were harvested from the 10-70% fractions. However, there were no living cells in the 70-80% fraction. We concluded that living OB-HipOPs are separated by one 10-70% Percoll gradient. Furthermore, we analyzed the expression patterns of putative MSC markers on differentiated HipOPs. FACS analysis revealed that Sca-1, CD44, CD73, CD105, and CD106 were decreased in OB-HipOPs. In adipogenic- and chondrogenic-HipOPs, Sca-1, CD73, CD105, and CD106 were decreased. This new technique is a helpful tool to identify MSC surface markers and to clarify in more detail the differentiation stages of osteoblasts.