Bone formation ability of Gli1+ cells in the periodontal ligament after tooth extraction.

During the process of socket healing after tooth extraction, osteoblasts appear in the tooth socket and form alveolar bone; however, the source of these osteoblasts is still uncertain. Recently, it has been demonstrated that cells expressing Gli1, a downstream factor of sonic hedgehog signaling, exhibit stem cell properties in the periodontal ligament (PDL). Therefore, in the present study, the differentiation ability of Gli1+-PDL cells after tooth extraction was analyzed using Gli1-CreERT2/ROSA26-loxP-stop-loxP-tdTomato (iGli1/Tomato) mice. After the final administration of tamoxifen to iGli1/Tomato mice, Gli1/Tomato+ cells were rarely detected in the PDL. One day after the tooth extraction, although inflammatory cells appeared in the tooth socket, Periostin+ PDL-like tissues having a few Gli1/Tomato+ cells remained near the alveolar bone. Three days after the extraction, the number of Gli1/Tomato+ cells increased as evidenced by numerous PCNA+ cells in the socket. Some of these Gli1/Tomato+ cells expressed BMP4 and Phosphorylated (P)-Smad1/5/8. After seven days, the Osteopontin+ bone matrix was formed in the tooth socket apart from the alveolar bone. Many Gli1/Tomato+ osteoblasts that were positive for Runx2+ were arranged on the surface of the newly formed bone matrix. In the absence of Gli1+-PDL cells in Gli1-CreERT2/Rosa26-loxP-stop-loxP-tdDTA (iGli1/DTA) mice, the amount of newly formed bone matrix was significantly reduced in the tooth socket. Therefore, these results collectively suggest that Gli1+-PDL cells differentiate into osteoblasts to form the bone matrix in the tooth socket; thus, this differentiation might be regulated, at least in part, by bone morphogenetic protein (BMP) signaling.

Immunohistochemical localization of CD146 and alpha-smooth muscle actin during dentin formation and regeneration.

Cluster of differentiation 146 (CD146) is known to localize in stem cells and precursor cells of various tissues. In this study, to analyze the function of CD146 in odontoblast differentiation, immunohistochemical localization of CD146 was examined during rat molar tooth development and after cavity preparation. At the cap and bell stages, many CD146-positive cells were visible around the blood vessels in the dental papillae. On Postnatal day 2, osterix-positive odontoblasts were arranged in the dentin sialoprotein (DSP)-positive predentin, and many CD146-positive cells were observed near these odontoblasts with blood vessels. Some perivascular CD146-positive cells overlapped with Smad4-positive cells. However, the immunoreactivity for alpha-smooth muscle actin (α-SMA), one of the markers for undifferentiated cells, was negligible. Furthermore, the number of these cells decreased in the dental pulp on Postnatal day 28. On Day 4 after cavity preparation, Osterix-positive odontoblasts appeared lining the reparative dentin. Most of the blood vessels near the reparative dentin showed immunoreactivities for CD146. Reparative odontoblasts actively formed DSP-positive dentin matrix because these cells were positive for Smad4 and Osterix, but not for α-SMA. After 7 days, the number of CD146-positive cells near blood vessels decreased in the dental pulp beneath the cavity. These results suggest that the CD146 is expressed in the perivascular area of the dental pulp and induces vascularization in the vicinity of dentin formation, and some CD146-positive cells are activated by the bone morphogenetic protein signaling pathway and differentiate into odontoblasts in the early stages of dentin formation and repair.

Immunohistochemical Analysis of CCN2 in Experimental Fracture Healing Models.

Skeletal fractures are most common large-organ traumatic injuries that impact the functions and esthetic outcomes and quality of life. Unfortunately, infection during the fracture healing process and inadequate blood supply to the bone impede reduced ability to produce cartilage and effective bone callus formation, leading to nonunion or delayed union fracture. Therefore, studying the mechanism of fracture healing is an important task in solving the problem of fracture healing failure. Animal models of bone fracture healing are important tools to investigate the pathogenesis and develop treatment strategies. This protocol introduces researchers to a bone repair model utilizing the ribs of rats and the immunohistological expression of cellular communication network factor/connective tissue growth factor (CTGF/CCN2) during the fracture healing processes.

Stem cell properties of Gli1-positive cells in the periodontal ligament.

BACKGROUND: The periodontal ligament (PDL), which surrounds the tooth root, contains mesenchymal stem cells (MSCs) capable of differentiating into osteoblasts, cementoblasts, and fibroblasts under normal conditions. These MSCs are thought to have important roles in the repair and regeneration of injured periodontal tissues. However, since there is no useful marker for MSCs in the PDL, the characteristics and distributions of these cells remain unclear. Gli1, an essential hedgehog signaling transcription factor, functions in undifferentiated cells during embryogenesis. Previous studies have demonstrated that the dental epithelial and mesenchymal cells positive for Gli1 in developing teeth have stem cell properties, including the ability to form colonies and pluripotency. Therefore, the focus of this review is the stem cell properties of Gli1-positive cells in the PDL, with an emphasis on the differentiation ability of osteoblasts for the regeneration of periodontal tissues. HIGHLIGHT: Lineage tracing analysis identified Gli1-positive PDL cells as MSCs that contribute to the formation of periodontal tissues and can regenerate alveolar bone. CONCLUSION: Gli1 is a potential stem cell marker in the PDL. A more definitive understanding of the functions of Gli1-positive cells could be useful for the development of regenerative methods using the MSCs in the PDL.

Expression and Role of IL-1ß Signaling in Chondrocytes Associated with Retinoid Signaling during Fracture Healing.

The process of fracture healing consists of an inflammatory reaction and cartilage and bone tissue reconstruction. The inflammatory cytokine interleukin-1ß (IL-1ß) signal is an important major factor in fracture healing, whereas its relevance to retinoid receptor (an RAR inverse agonist, which promotes endochondral bone formation) remains unclear. Herein, we investigated the expressions of IL-1ß and retinoic acid receptor gamma (RARγ) in a rat fracture model and the effects of IL-1ß in the presence of one of several RAR inverse agonists on chondrocytes. An immunohistochemical analysis revealed that IL-1ß and RARγ were expressed in chondrocytes at the fracture site in the rat ribs on day 7 post-fracture. In chondrogenic ATDC5 cells, IL-1ß decreases the levels of aggrecan and type II collagen but significantly increased the metalloproteinase-13 (Mmp13) mRNA by real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis. An RAR inverse agonist (AGN194310) inhibited IL-1ß-stimulated Mmp13 and Ccn2 mRNA in a dose-dependent manner. Phosphorylated extracellular signal regulated-kinases (pERK1/2) and p-p38 mitogen-activated protein kinase (MAPK) were increased time-dependently by IL-1ß treatment, and the IL-1ß-induced p-p38 MAPK was inhibited by AGN194310. Experimental p38 inhibition led to a drop in the IL-1ß-stimulated expressions of Mmp13 and Ccn2 mRNA. MMP13, CCN2, and p-p38 MAPK were expressed in hypertrophic chondrocytes near the invaded vascular endothelial cells. As a whole, these results point to role of the IL-1ß via p38 MAPK as important signaling in the regulation of the endochondral bone formation in fracture healing, and to the actions of RAR inverse agonists as potentially relevant modulators of this process.

Silkworms suppress the release of green leaf volatiles by mulberry leaves with an enzyme from their spinnerets.

In response to herbivory, plants emit a blend of volatile organic compounds that includes green leaf volatiles (GLVs) and terpenoids. These volatiles are known to attract natural enemies of herbivores and are therefore considered to function as an indirect defense. Selection should favor herbivores that are able to suppress these volatile emissions, and thereby make themselves less conspicuous to natural enemies. We tested this possibility for silkworms, which were observed to leave secretions from their spinnerets while feeding on mulberry leaves. When we ablated the spinnerets of silkworms, no secretions were observed. Leaves infested by intact silkworms released smaller amounts of GLVs than leaves infested by ablated silkworms, indicating that the spinneret secretion suppressed GLV production. This difference in GLV emissions was also reflected in the behavioral response of Zenillia dolosa (Tachinidae), a parasitoid fly of silkworms. The flies laid fewer eggs when exposed to the volatiles from intact silkworm-infested leaves than when exposed to the volatiles from ablated silkworm-infested leaves. We identified a novel enzyme in the secretion from the spinneret that is responsible for the GLV suppression. The enzyme converted 13(S)-hydroperoxy-(9Z,11E,15Z)-octadecatrienoic acid, an intermediate in the biosynthetic pathway of GLVs, into its keto-derivative in a stereospecific manner. Taken together, this study shows that silkworms are able to feed on mulberry in a stealthy manner by suppressing GLV production with an enzyme in secretions of their spinnerets, which might be a countermeasure against induced indirect defense by mulberry plants.

Cooperative Polar/Steric Strategy in Achieving Site-Selective Photocatalyzed C(sp3 )-H Functionalization.

Synergistic control over the SH 2 transition states of hydrogen abstraction exploiting polar and steric effects provides a promising cooperative strategy for site-selective C(sp3 )-H functionalization using decatungstate anion photocatalysis. By using this photocatalytic approach, the C-H bonds of substituted lactones and cyclic ketones were functionalized selectively. In the remarkable case of 2-isoamyl 4-tert-butyl cyclohexanone (1 t) bearing five methyl, five methylene, and three methine C-H bonds, one methine C-H bond in the isoamyl tether was selectively functionalized.

Versatile cross-dehydrogenative coupling of heteroaromatics and hydrogen donors via decatungstate photocatalysis.

A facile sunlight-induced derivatization of heteroaromatics via photocatalyzed C-H functionalization in amides, ethers, alkanes and aldehydes is described. Tetrabutylammonium decatungstate (TBADT) was used as the photocatalyst and allowed to carry out the process under mild conditions.