Taxonomic difference in marine bloom-forming phytoplanktonic species affects the dynamics of both bloom-responding prokaryotes and prokaryotic viruses.

The production of dissolved organic matter during phytoplankton blooms and consumption by heterotrophic prokaryotes promote marine carbon biogeochemical cycling. Although prokaryotic viruses presumably affect this process, their dynamics during blooms are not fully understood. Here, we investigated the effects of taxonomic difference in bloom-forming phytoplankton on prokaryotes and their viruses. We analyzed the dynamics of coastal prokaryotic communities and viruses under the addition of dissolved intracellular fractions from taxonomically distinct phytoplankton, the diatom Chaetoceros sp. (CIF) and the raphidophycean alga Heterosigma akashiwo (HIF), using microcosm experiments. Ribosomal RNA gene amplicon and viral metagenomic analyses revealed that particular prokaryotes and prokaryotic viruses specifically increased in either CIF or HIF, indicating that taxonomic difference in bloom-forming phytoplankton promotes distinct dynamics of not only the prokaryotic community but also prokaryotic viruses. Furthermore, combining our microcosm experiments with publicly available environmental data mining, we identified both known and novel possible host-virus pairs. In particular, the growth of prokaryotes associating with phytoplanktonic organic matter, such as Bacteroidetes (Polaribacter and NS9 marine group), Vibrio spp., and Rhodobacteriales (Nereida and Planktomarina), was accompanied by an increase in viruses predicted to infect Bacteroidetes, Vibrio, and Rhodobacteriales, respectively. Collectively, our findings suggest that changes in bloom-forming species can be followed by an increase in a specific group of prokaryotes and their viruses and that elucidating these tripartite relationships among specific phytoplankton, prokaryotes, and prokaryotic viruses improves our understanding of coastal biogeochemical cycling in blooms.IMPORTANCEThe primary production during marine phytoplankton bloom and the consumption of the produced organic matter by heterotrophic prokaryotes significantly contribute to coastal biogeochemical cycles. While the activities of those heterotrophic prokaryotes are presumably affected by viral infection, the dynamics of their viruses during blooms are not fully understood. In this study, we experimentally demonstrated that intracellular fractions of taxonomically distinct bloom-forming phytoplankton species, the diatom Chaetoceros sp. and the raphidophycean alga Heterosigma akashiwo, promoted the growth of taxonomically different prokaryotes and prokaryotic viruses. Based on their dynamics and predicted hosts of those viruses, we succeeded in detecting already-known and novel possible host-virus pairs associating with either phytoplankton species. Altogether, we propose that the succession of bloom-forming phytoplankton would change the composition of the abundant prokaryotes, resulting in an increase in their viruses. These changes in viral composition, depending on bloom-forming species, would alter the dynamics and metabolism of prokaryotes, affecting biogeochemical cycling in blooms.

Osteoblast differentiation of Gli1⁺ cells via Wnt and BMP signaling pathways during orthodontic tooth movement.

OBJECTIVES: Factors that induce bone formation during orthodontic tooth movement (OTM) remain unclear. Gli1 was recently identified as a stem cell marker in the periodontal ligament (PDL). Therefore, we evaluated the mechanism of differentiation of Cre/LoxP-mediated Gli1/Tomato+ cells into osteoblasts during OTM. METHODS: After the final administration of tamoxifen to 8-week-old Gli1-CreERT2/ROSA26-loxP-stop-loxP-tdTomato mice for 2 days, nickel-titanium closed coil springs were attached between the upper anterior alveolar bone and the first molar. Immunohistochemical localizations of ß-catenin, Smad4, and Runx2 were observed in the PDL on 2, 5, and 10 days after OTM initiation. RESULTS: In the untreated tooth, few Gli1/Tomato+ cells were detected in the PDL. Two days after OTM initiation, the number of Gli1/Tomato+ cells increased in the PDL on the tension side. On this side, 49.3 ±â€¯7.0% of ß-catenin+ and 48.7 ±â€¯5.7% of Smad4+ cells were found in the PDL, and Runx2 expression was detected in some Gli1/Tomato+ cells apart from the alveolar bone. The number of positive cells in the PDL reached a maximum on day 5. In contrast, on the compression side, ß-catenin and Smad4 exhibited less immunoreactivity. On day 10, Gli1/Tomato+ cells were aligned on the alveolar bone on the tension side, with some expressing Runx2. CONCLUSIONS: Gli1+ cells in the PDL differentiated into osteoblasts during OTM. Wnt and bone morphogenetic proteins signaling pathways may be involved in this differentiation.

A rumen virosphere with implications of contribution to fermentation and methane production, and endemism in cattle breeds and individuals.

Viruses have a potential to modify the ruminal digestion via infection and cell lysis of prokaryotes, suggesting that viruses are related to animal performance and methane production. This study aimed to elucidate the genome-based diversity of rumen viral communities and the differences in virus structure between individuals and cattle breeds and to understand how viruses influence on the rumen. To these ends, a metagenomic sequencing of virus-like particles in the rumen of 22 Japanese cattle, including Japanese Black (JB, n = 8), Japanese Shorthorn (n = 2), and Japanese Black sires × Holstein dams crossbred steers (F1, n = 12) was conducted. Additionally, the rumen viromes of six JB and six F1 that were fed identical diets and kept in a single barn were compared. A total of 8,232 non-redundant viral genomes (≥5-kb length and ≥50% completeness), including 982 complete genomes, were constructed, and rumen virome exhibited lysogenic signatures. Furthermore, putative hosts of 1,223 viral genomes were predicted using tRNA and clustered regularly interspaced short palindromic repeat (CRISPR)-spacer matching. The genomes included 1 and 10 putative novel complete genomes associated with Fibrobacter and Ruminococcus, respectively, which are the main rumen cellulose-degrading bacteria. Additionally, the hosts of 22 viral genomes, including 2 complete genomes, were predicted as methanogens, such as Methanobrevibacter and Methanomethylophilus. Most rumen viruses were highly rumen and individual specific and related to rumen-specific prokaryotes. Furthermore, the rumen viral community structure was significantly different between JB and F1 steers, indicating that cattle breed is one of the factors influencing the rumen virome composition.IMPORTANCEHere, we investigated the individual and breed differences of the rumen viral community in Japanese cattle. In the process, we reconstructed putative novel complete viral genomes related to rumen fiber-degrading bacteria and methanogen. The finding strongly suggests that rumen viruses contribute to cellulose and hemicellulose digestion and methanogenesis. Notably, this study also found that rumen viruses are highly rumen and individual specific, suggesting that rumen viruses may not be transmitted through environmental exposure. More importantly, we revealed differences of viral communities between JB and F1 cattle, indicating that cattle breed is a factor that influences the establishment of rumen virome. These results suggest the possibility of rumen virus transmission from mother to offspring and its potential to influence beef production traits. These rumen viral genomes and findings provide new insights into the characterizations of the rumen viruses.

Population-level prokaryotic community structures associated with ferromanganese nodules in the Clarion-Clipperton Zone (Pacific Ocean) revealed by 16S rRNA gene amplicon sequencing.

Although deep-sea ferromanganese nodules are a potential resource for exploitation, their formation mechanisms remain unclear. Several nodule-associated prokaryotic species have been identified by amplicon sequencing of 16S rRNA genes and are assumed to contribute to nodule formation. However, the recent development of amplicon sequence variant (ASV)-level monitoring revealed that closely related prokaryotic populations within an operational taxonomic unit often exhibit distinct ecological properties. Thus, conventional species-level monitoring might have overlooked nodule-specific populations when distinct populations of the same species were present in surrounding environments. Herein, we examined the prokaryotic community diversity of nodules and surrounding environments at the Clarion-Clipperton Zone in Japanese licensed areas by 16S rRNA gene amplicon sequencing with ASV-level resolution for three cruises from 2017 to 2019. Prokaryotic community composition and diversity were distinct by habitat type: nodule, nodule-surface mud, sediment, bottom water and water column. Most ASVs (~80%) were habitat-specific. We identified 178 nodule-associated ASVs and 41 ASVs associated with nodule-surface mud via linear discriminant effect size analysis. Moreover, several ASVs, such as members of SAR324 and Woeseia, were highly specific to nodules. These nodule-specific ASVs are promising targets for future investigation of the nodule formation process.

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.

The Role of Hedgehog Signaling in the Melanoma Tumor Bone Microenvironment.

A crucial regulator in melanoma progression and treatment resistance is tumor microenvironments, and Hedgehog (Hh) signals activated in a tumor bone microenvironment are a potential new therapeutic target. The mechanism of bone destruction by melanomas involving Hh/Gli signaling in such a tumor microenvironment is unknown. Here, we analyzed surgically resected oral malignant melanoma specimens and observed that Sonic Hedgehog, Gli1, and Gli2 were highly expressed in tumor cells, vasculatures, and osteoclasts. We established a tumor bone destruction mouse model by inoculating B16 cells into the bone marrow space of the right tibial metaphysis of 5-week-old female C57BL mice. An intraperitoneal administration of GANT61 (40 mg/kg), a small-molecule inhibitor of Gli1 and Gli2, resulted in significant inhibition of cortical bone destruction, TRAP-positive osteoclasts within the cortical bone, and endomucin-positive tumor vessels. The gene set enrichment analysis suggested that genes involved in apoptosis, angiogenesis, and the PD-L1 expression pathway in cancer were significantly altered by the GANT61 treatment. A flow cytometry analysis revealed that PD-L1 expression was significantly decreased in cells in which late apoptosis was induced by the GANT61 treatment. These results suggest that molecular targeting of Gli1 and Gli2 may release immunosuppression of the tumor bone microenvironment through normalization of abnormal angiogenesis and bone remodeling in advanced melanoma with jaw bone invasion.

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.

Differentiation ability of Gli1+ cells during orthodontic tooth movement.

Orthodontic tooth movement (OTM) induces bone formation on the alveolar bone of the tension side; however, the mechanism of osteoblast differentiation is not fully understood. Gli1 is an essential transcription factor for hedgehog signaling and functions in undifferentiated cells during embryogenesis. In this study, we examined the differentiation of Gli1+ cells in the periodontal ligament (PDL) during OTM using a lineage-tracing analysis. After the final administration of tamoxifen for 2 days to 8-week-old Gli1-CreERT2/ROSA26-loxP-stop-loxP-tdTomato (iGli1/Tomato) mice, Gli1/Tomato+ cells were rarely observed near endomucin+ blood vessels in the PDL. Osteoblasts lining the alveolar bone did not exhibit Gli1/Tomato fluorescence. To move the first molar of iGli1/Tomato mice medially, nickel-titanium closed-coil springs were attached between the upper anterior alveolar bone and the first molar. Two days after OTM initiation, the number of Gli1/Tomato+ cells increased along with numerous PCNA+ cells in the PDL of the tension side. As some Gli1/Tomato+ cells exhibited positive expression of osterix, an osteoblast differentiation marker, Gli1+ cells probably differentiated into osteoblast progenitor cells. On day 10, the newly formed bone labeled by calcein administration during OTM was detected on the surface of the original alveolar bone of the tension side. Gli1/Tomato+ cells expressing osterix localized to the surface of the newly formed bone. In contrast, in the PDL of the compression side, Gli1/Tomato+ cells proliferated before day 10 and expressed type I collagen, suggesting that the Gli1+ cells also differentiated into fibroblasts. Collectively, these results demonstrate that Gli1+ cells in the PDL can differentiate into osteoblasts at the tension side and may function in bone remodeling as well as fibril formation in the PDL during OTM.

Human dentin materials for minimally invasive bone regeneration: Animal studies and clinical cases.

OBJECTIVES: Bone, platelet concentrate, and tooth-derived dentin/cementum have been used as autologous materials in regenerative medicine Dentin materials were first recycled in 2002 for bone regeneration in humans, although bone autografts were noted in the 19th century, and auto-platelet concentrates were developed in 1998. Dentin/cementum-based material therapy has been applied as an innovative technique for minimally invasive bone surgery, while bone autografts are associated with donor site morbidity. METHODS: In October 2021, PubMed, Google Scholar, Scopus, and the Cochrane Library databases from 1980 to 2020 were screened. RESULTS: The demineralized dentin/cementum matrix (DDM) had better performance in bone induction and bone regeneration than mineralized dentin. CONCLUSIONS: Unlike cell culture therapy, DDM is a matrix-based therapy that includes growth factors. A matrix-based system is a realistic and acceptable treatment, even in developing countries. The aim of this review was to summarize the evidence related to both animal studies and human clinical cases using human dentin materials with a patch of cementum, especially DDM.

Identification of 146 Metagenome-assembled Genomes from the Rumen Microbiome of Cattle in Japan.

The rumen contains a complex microbial ecosystem that degrades plant materials, such as cellulose and hemicellulose. We herein reconstructed 146 nonredundant, rumen-specific metagenome-assembled genomes (MAGs), with ≥50% completeness and <10% contamination, from cattle in Japan. The majority of MAGs were potentially novel strains, encoding various enzymes related to plant biomass degradation and volatile fatty acid production. The MAGs identified in the present study may be valuable resources to enhance the resolution of future taxonomical and functional studies based on metagenomes and metatranscriptomes.

Localization of Bmi1 in osteoblast-lineage cells during endochondral ossification.

Encoded by B cell-specific moloney murine leukemia virus integration site 1, Bmi1 is part of the polycomb group of proteins localized in stem and undifferentiated cells. It regulates the expression of various differentiation genes. However, the regulatory mechanism of skeletal development by Bmi1 remains poorly understood. In this study, we aimed to observe Bmi1 distribution during endochondral ossification processes in rat bone development and fracture healing. Immunoreactivity of Bmi1 was detected in the mesenchymal cell aggregation area at embryonic day (E) 14 and in cells around the center of cartilage primordium at E 16. Subsequently, the calcified bone matrix was formed around the cartilage primordium, and osteoblasts expressing Runt-related transcription factor 2 (Runx2) and Osterix (Osx) showed immunopositivity for Bmi1. At 4 days after bone fracture, the connective tissue around the fractured bone contained Bmi1-positive cells. At 42 days after fracture, osteoblasts along the surface of the new bone revealed Bmi1-, Runx2- and Osx-positive reactions, but the Bmi1 immunoreactivity in osteocytes was less than the Runx2 and Osx immunoreactivities. In conclusion, Bmi1 is localized in the osteoblast-lineage cells in their early differentiation stages, and it might regulate their differentiation during endochondral ossification.

Taxonomic and functional characterization of the rumen microbiome of Japanese Black cattle revealed by 16S rRNA gene amplicon and metagenome shotgun sequencing.

This study aimed to determine the taxonomic and functional characteristics of the Japanese Black (JB) steer rumen microbiome. The rumen microbiomes of six JB steers (age 14.7 ± 1.44 months) and six JB sires × Holstein dams crossbred (F1) steers (age 11.1 ± 0.39 months), fed the same diet, were evaluated. Based on 16S rRNA gene sequencing, the beta diversity revealed differences in microbial community structures between the JB and F1 rumen. Shotgun sequencing showed that Fibrobacter succinogenes and two Ruminococcus spp., which are related to cellulose degradation were relatively more abundant in the JB steer rumen than in the F1 rumen. Furthermore, the 16S rRNA gene copy number of F. succinogenes was significantly higher in the JB steer rumen than in the F1 rumen according to quantitative real-time polymerase chain reaction analysis. Genes encoding the enzymes that accelerate cellulose degradation and those associated with hemicellulose degradation were enriched in the JB steer rumen. Although Prevotella spp. were predominant both in the JB and F1 rumen, the genes encoding carbohydrate-active enzymes of Prevotella spp. may differ between JB and F1.

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.

Differential Responses of a Coastal Prokaryotic Community to Phytoplanktonic Organic Matter Derived from Cellular Components and Exudates.

The phytoplanktonic production and prokaryotic consumption of organic matter significantly contribute to marine carbon cycling. Organic matter released from phytoplankton via three processes (exudation of living cells, cell disruption through grazing, and viral lysis) shows distinct chemical properties. We herein investigated the effects of phytoplanktonic whole-cell fractions (WF) (representing cell disruption by grazing) and extracellular fractions (EF) (representing exudates) prepared from Heterosigma akashiwo, a bloom-forming Raphidophyceae, on prokaryotic communities using culture-based experiments. We analyzed prokaryotic community changes for two weeks. The shift in cell abundance by both treatments showed similar dynamics, reaching the first peak (~4.1×106| |cells| |mL-1) on day 3 and second peak (~1.1×106| |cells| |mL-1) on day 13. We classified the sequences obtained into operational taxonomic units (OTUs). A Bray-Curtis dissimilarity analysis revealed that the OTU-level community structure changed distinctively with the two treatments. Ten and 13 OTUs were specifically abundant in the WF and EF treatments, respectively. These OTUs were assigned as heterotrophic bacteria mainly belonging to the Alteromonadales (Gammaproteobacteria) and Bacteroidetes clades and showed successive dynamics following the addition of organic matter. We also analyzed the dynamics of these OTUs in the ocean using publicly available metagenomic data from a natural coastal bloom in Monterey Bay, USA. At least two WF treatment OTUs showed co-occurrence with H. akashiwo, indicating that the blooms of H. akashiwo also affect these OTUs in the ocean. The present results strongly suggest that the thriving and dead cells of uninfected phytoplankton differentially influence the marine prokaryotic community.

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.

Sonic Hedgehog Signaling and Tooth Development.

Sonic hedgehog (Shh) is a secreted protein with important roles in mammalian embryogenesis. During tooth development, Shh is primarily expressed in the dental epithelium, from initiation to the root formation stages. A number of studies have analyzed the function of Shh signaling at different stages of tooth development and have revealed that Shh signaling regulates the formation of various tooth components, including enamel, dentin, cementum, and other soft tissues. In addition, dental mesenchymal cells positive for Gli1, a downstream transcription factor of Shh signaling, have been found to have stem cell properties, including multipotency and the ability to self-renew. Indeed, Gli1-positive cells in mature teeth appear to contribute to the regeneration of dental pulp and periodontal tissues. In this review, we provide an overview of recent advances related to the role of Shh signaling in tooth development, as well as the contribution of this pathway to tooth homeostasis and regeneration.

Sonic Hedgehog Regulates Bone Fracture Healing.

Bone fracture healing involves the combination of intramembranous and endochondral ossification. It is known that Indian hedgehog (Ihh) promotes chondrogenesis during fracture healing. Meanwhile, Sonic hedgehog (Shh), which is involved in ontogeny, has been reported to be involved in fracture healing, but the details had not been clarified. In this study, we demonstrated that Shh participated in fracture healing. Six-week-old Sprague-Dawley rats and Gli-CreERT2; tdTomato mice were used in this study. The right rib bones of experimental animals were fractured. The localization of Shh and Gli1 during fracture healing was examined. The localization of Gli1 progeny cells and osterix (Osx)-positive cells was similar during fracture healing. Runt-related transcription factor 2 (Runx2) and Osx, both of which are osteoblast markers, were observed on the surface of the new bone matrix and chondrocytes on day seven after fracture. Shh and Gli1 were co-localized with Runx2 and Osx. These findings suggest that Shh is involved in intramembranous and endochondral ossification during fracture healing.

Boric acid inhibits alveolar bone loss in rat experimental periodontitis through diminished bone resorption and enhanced osteoblast formation.

BACKGROUND/PURPOSE: Inhibition of bone resorption is essential for periodontal treatment. Recently, it has been suggested that boric acid suppresses periodontitis, but the mechanism of this inhibition is still not well understood. Therefore, to analyze the cellular response to boric acid administration, we histologically evaluated alveolar bone in experimental periodontitis of rats administered boric acid. MATERIALS AND METHODS: 5-0 silk ligatures were placed around the cervix of the second maxillary molars of 4 week-old rats treated with or without boric acid. Five and 14 days after ligature placement, the periodontal tissues between first and second molars were investigated histologically and immunohistochemically using antibodies to CD68, cathepsin K, and α-smooth muscle actin (SMA). RESULTS: Five days after the beginning of the experiment, many CD68-positive cells appeared in the periodontal tissues with ligature placement without boric acid administration. Also, the number of cathepsin K-positive osteoclasts had increased on the surface of alveolar bone. However, boric acid administration prevented severe bone resorption and reduced the number of cells positive for CD68 and cathepsin K. At day 14 post treatment, cells positive for α-SMA were seen in the periodontal tissues after boric acid administration, whereas no such cells were found around the alveolar bone without the administration of boric acid. CONCLUSION: Boric acid inhibited the inflammation of ligature-induced periodontitis. This agent might reduce bone resorption by inhibiting osteoclastogenesis and also could accelerate osteoblastogenesis.

Effects of tooth storage media on periodontal ligament preservation.

BACKGROUND/AIMS: An easily available tooth storage medium is required to preserve a tooth after avulsion. Milk and Hank's balanced salt solution (HBSS) are recommended as tooth storage media, and egg white is also reported to be comparable with milk. The aim of this study was to histologically and immunohistochemically evaluate the effect of different tooth storage media on the periodontal ligament (PDL) of extracted teeth. MATERIALS AND METHODS: This experiment used HBSS, milk, and egg white as tooth storage media. A total of ninety-six 6-week-old male Sprague-Dawley rats were used in these experiments. In each experiment, six rats were used for each medium and for the control group. Extracted rat molar teeth were immersed in these three different storage media for 1 hour. In each medium, six samples (n=18) were fixed immediately, and the remaining samples (n=54) were subcutaneously transplanted. In the control group (n=24), the extracted teeth were fixed or transplanted immediately after extraction. At day 4, 1 and 2 weeks after transplantation, the teeth were examined by radiographic, histological, and immunohistochemical methods. The number of PDL cells in the storage media was also counted. RESULTS: Teeth immersed for 1 hour in milk showed the thinnest PDL. Immunohistochemistry of periostin and CD68 labeling suggested degradation of the extracellular matrix in the PDL. In the media used for immersion, more PDL cells were observed in milk than in the other solutions. After transplantation, the HBSS and egg white groups maintained adequate thickness of PDL but in the milk group, thinner PDL and ankylosis were observed. CONCLUSION: Adequate thickness of PDL was maintained in the egg white group, whereas the milk group showed disturbance in the PDL, which may lead to ankylosis.