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.

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.

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.

Minodronic acid induces morphological changes in osteoclasts at bone resorption sites and reaches a level required for antagonism of purinergic P2X2/3 receptors.

Minodronic acid is an aminobisphosphonate that is an antagonist of purinergic P2X2/3 receptors involved in pain. The aim of this study was to investigate the action and distribution of minodronic acid and the potential for P2X2/3 receptor antagonism based on the estimated concentration of minodronic acid. Microlocalization of radiolabeled minodronic acid was examined in the femur of neonatal rats. The bone-binding characteristics of minodronic acid and morphological changes in osteoclasts were analyzed in vitro. The minodronic acid concentration around bone resorption lacunae was predicted based on bone binding and the shape of lacunae. In microautoradiography, radioactive silver grains were abundant in bone-attached osteoclasts and were detected in calcified and ossification zones and in the cytoplasm of osteoclasts but not in the hypertrophic cartilage zone. In an osteoclast culture with 1 µM minodronic acid, 65% of minodronic acid was bound to bone, and C-terminal cross-linking telopeptide release was inhibited by 96%. Cultured osteoclasts without minodronic acid treatment formed ruffled borders and bone resorption lacunae and had rich cytoplasm, whereas those treated with 1 µM minodronic acid were not multinucleated, stained densely with toluidine blue, and were detached from the bone surface. In the 1 µM culture, the estimated minodronic acid concentration in resorption lacunae was 880 µM, which is higher than the IC50 for minodronic acid antagonism of P2X2/3 receptors. Thus, inhibition of P2X2/3 receptors around osteoclasts may contribute to the analgesic effect of minodronic acid.

Expression and localization of CRAMP in rat tooth germ and during reparative dentin formation.

OBJECTIVES: Cathelicidin-related antimicrobial peptide (CRAMP) is an antimicrobial peptide in mice and rats homologous to LL-37 in humans. In addition to its antibacterial activity, CRAMP has various physiological functions by binding to formyl peptide receptor 2 (FPR2). However, the role of these peptides in teeth is unknown. Therefore, we investigated the role of CRAMP and FPR2 in tooth development, reparative dentin formation, and defense response. MATERIAL AND METHODS: First, we examined the localization of CRAMP and FPR2 during tooth development by immunohistochemical analysis. Next, we investigated the localization of CRAMP, FPR2, and CD68-positive macrophages by immunohistochemical analysis during pulp inflammation and reparative dentin formation after cavity preparation. Finally, we analyzed the effect of lipopolysaccharide (LPS) on the expression of CRAMP and FPR2 in dental pulp cells by real-time reverse transcription PCR. RESULTS: At the late bell stage in tooth development, CRAMP was detected in odontoblasts, and FPR2 was observed in the sub-odontoblastic layer. In mature teeth, CRAMP was not detected, but FPR2 continued to be localized in the sub-odontoblastic layer. After cavity preparation, CRAMP-positive cells and macrophages were found in dental pulp tissues below the cavity at an early stage of repair. At subsequent stages of reparative dentin formation, CRAMP was observed in odontoblast-like cells that contacted reparative dentin. FPR2 immunoreactivity was also detected in odontoblast-like cells and neighboring cells. LPS stimulated the expression of CRAMP mRNA in dental pulp cells in vitro. CONCLUSIONS: Localization of CRAMP and its receptor FPR2-positive cells were observed during physiological and reparative dentin formation. CLINICAL RELEVANCE: CRAMP/LL-37 has a possibility that induce reparative dentin formation.

The molecular chaperone Hsp47 is essential for cartilage and endochondral bone formation.

Heat shock protein 47 kDa (Hsp47) is considered as a molecular chaperone essential for the correct folding of type I and type IV procollagen in the ER. However, the function of Hsp47 for other types of procollagen and its importance for chondrogenesis have never been elucidated. To examine the function of Hsp47 in cartilage formation and endochondral ossification, we conditionally inactivated the Hsp47 gene in chondrocytes using Hsp47 floxed mice and mice carrying a chondrocyte-specific Col2a1-Cre transgene. Hsp47 conditional null mutant mice died just before or shortly after birth, and exhibited severe generalized chondrodysplasia and bone deformities with lower levels of type II and type XI collagen. Second-harmonic generation (SHG) analysis and electron microscopy revealed the accumulation of misaligned type I collagen molecules in the intervertebral discs and a substantial decrease in type II collagen fibers, respectively. Whole-mount skeletal staining showed no calcified region in the vertebral bodies of sacral vertebrae, and revealed that the endochondral bones were severely twisted and shortened. These results demonstrate that Hsp47 is indispensable for well-organized cartilage and normal endochondral bone formation.

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 ± 7.0% of ß-catenin+ and 48.7 ± 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.

Localization of SUMOylation factors and Osterix in odontoblast lineage cells during dentin formation and regeneration.

Small ubiquitin-related modifier (SUMO) conjugation (SUMOylation) is a post-translational modification involved in various cellular processes including the regulation of transcription factors. In this study, to analyze the involvement of SUMOylation in odontoblast differentiation, we examined the immunohistochemical localization of SUMO-1, SUMO-2/3, and Osterix during rat tooth development. At the bud and cap stages, localization of SUMOs and Osterix was hardly detected in the dental mesenchyme. At the bell stage, odontoblasts just beginning dentin matrix secretion and preodontoblasts near these odontoblasts showed intense immunoreactivity for these molecules. However, after the root-formation stage, these immunoreactivities in the odontoblasts decreased in intensity. Next, to examine whether the SUMOylation participates in dentin regeneration, we evaluated the distribution of SUMOs and Osterix in the dental pulp after cavity preparation. In the coronal pulp chamber of an untreated rat molar, odontoblasts and pulp cells showed no immunoreactivity. At 4 days after cavity preparation, positive cells for SUMOs and Osterix appeared on the surface of the dentin beneath the cavity. Odontoblast-like cells forming reparative dentin were immunopositive for SUMOs and Osterix at 1 week, whereas these immunoreactivities disappeared after 8 weeks. Additionally, we further analyzed the capacity of SUMO-1 to bind Osterix by performing an immunoprecipitation assay using C2C12 cells, and showed that Osterix could undergo SUMOylation. These results suggest that SUMOylation might regulate the transcriptional activity of Osterix in odontoblast lineage cells, and thus play important roles in odontoblast differentiation and regeneration.

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.

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.

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.

Immunohistochemical analysis of two stem cell markers of α-smooth muscle actin and STRO-1 during wound healing of human dental pulp.

Recent studies have employed two markers, alpha-smooth muscle actin (α-SMA) and STRO-1, to detect cells with mesenchymal stem cell properties in dental pulp. The present study aimed to explore the expression profile of α-SMA and STRO-1 in intact dental pulp as well as during wound healing in adult dental pulp tissue. Healthy pulps were mechanically exposed and capped with the clinically used materials MTA (ProRoot White MTA) or Ca(OH)2 to induce a mineralized barrier at the exposed surface. After 7-42 days, the teeth were extracted and processed for immunohistochemical analysis using antibodies against α-SMA, STRO-1 and nestin (a neurogenic cytoskeletal protein expressed in odontoblasts). In normal pulp, α-SMA was detected in vascular smooth muscle cells and pericytes. Double immunofluorescent staining with STRO-1 and α-SMA showed that STRO-1 was localized in vascular smooth muscle cells, pericytes and endothelial cells, in addition to nerve fibers. During the process of dental pulp healing, numerous α-SMA-positive cells emerged at the wound margin at 14 days, and the initially formed mineralized barrier was lined with α-SMA-positive cells similar in appearance to reparative odontoblasts, some of which co-expressed nestin. STRO-1 was abundant in nerve fibers. In the advanced stage of mineralized barrier formation at 42 days, cells lining the barrier were stained with nestin, and no staining of α-SMA was detected in those cells. These observations indicate that α-SMA-positive cells temporarily appear along the wound margin during the earlier phase of mineralized barrier formation and STRO-1 is confined in vascular and neuronal elements.

Thy-1-positive cells in the subodontoblastic layer possess high potential to differentiate into hard tissue-forming cells.

The cells of the subodontoblastic cell-rich layer in dental pulp are speculated to contain odontoblast progenitor cells because of their positional relationship with odontoblasts as well as their high alkaline phosphatase (ALP) activity. However, it has yet to be determined whether these cells have the ability to differentiate into odontoblastic cells. In the present study, we firstly found that the majority of cells in the subodontoblastic layer expressed Thy-1, a cell-surface marker of stem and progenitor cells. Then, we evaluated the capacity of Thy-1 high- and low-expressing (Thy-1(high) and Thy-1(low)) cells separated from rat dental pulp cells by use of a fluorescence-activated cell sorter to differentiate into hard tissue-forming cells in vitro and in vivo. Following stimulation with bone morphogenetic protein-2, Thy-1(high) cells in vitro showed accelerated induction of ALP activity and formation of alizarin red-positive mineralized matrix compared with Thy-1(low) cells. Furthermore, subcutaneous implantation of Thy-1(high) cells efficiently induced the formation of bone-like matrix. These results collectively suggest that Thy-1-positive dental pulp cells localized in the subodontoblastic layer had the ability to differentiate into hard tissue-forming cells, and thus these cells may serve as a source of odontoblastic cells.

Runx3 is a crucial regulator of alveolar differentiation and lung tumorigenesis in mice.

The runt-domain transcription factor Runx3 plays crucial roles during development such as regulating gene expression. It has been shown that Runx3 is involved in neurogenesis, thymopoiesis and functions like a tumor suppressor. Runx3 null mouse die soon after birth as a result of multiple organ defects. Runx3 null mouse lung shows an abnormal phenotype and loss of Runx3 induced remodeling in the lung. Interestingly, lung adenocarcinoma is observed in Runx3 heterozygous mice at 18 months of age. During lung development various cellular and molecular events occur such as cell proliferation, cell death, differentiation and epithelial-mesenchymal transition (EMT). To understand the specific lethal events in Runx3 null mice, we examined cellular and molecular networks involved in EMT, and EMT inducers were quantified by RT-qPCR during lung development. Excessive EMT was observed in lungs at PN1 day in Runx3 null mice and PN18 months in Runx3 heterozygous mice. Pharmacologic inhibition of EMT was used to curb tumor progression. In this study, U0126 was injected to pregnant mouse for inhibition of pERK signaling. After U0126 treatment, life spans of newborn mice were increased and lung hyperplasia was partially rescued by down-regulated cell proliferation and EMT. Our data suggest that Runx3 is involved in crucial regulation of alveolar differentiation and tumor suppression in developing mouse lung.

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.

RANKL/OPG ratio regulates odontoclastogenesis in damaged dental pulp.

Bone-resorbing osteoclasts are regulated by the relative ratio of the differentiation factor, receptor activator NF-kappa B ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG). Dental tissue-localized-resorbing cells called odontoclasts have regulatory factors considered as identical to those of osteoclasts; however, it is still unclear whether the RANKL/OPG ratio is a key factor for odontoclast regulation in dental pulp. Here, we showed that odontoclast regulators, macrophage colony-stimulating factor-1, RANKL, and OPG were detectable in mouse pulp of molars, but OPG was dominantly expressed. High OPG expression was expected to have a negative regulatory effect on odontoclastogenesis; however, odontoclasts were not detected in the dental pulp of OPG-deficient (KO) mice. In contrast, damage induced odontoclast-like cells were seen in wild-type pulp tissues, with their number significantly increased in OPG-KO mice. Relative ratio of RANKL/OPG in the damaged pulp was significantly higher than in undamaged control pulp. Pulp damages enhanced hypoxia inducible factor-1α and -2α, reported to increase RANKL or decrease OPG. These results reveal that the relative ratio of RANKL/OPG is significant to pulpal odontoclastogenesis, and that OPG expression is not required for maintenance of pulp homeostasis, but protects pulp from odontoclastogenesis caused by damages.

Odontoblast death drives cell-rich zone-derived dental tissue regeneration.

Severe dental tissue damage induces odontoblast death, after which dental pulp stem and progenitor cells (DPSCs) differentiate into odontoblast-like cells, contributing to reparative dentin. However, the damage-induced mechanism that triggers this regeneration process is still not clear. We aimed to understand the effect of odontoblast death without hard tissue damage on dental regeneration. Herein, using a Cre/LoxP-based strategy, we demonstrated that cell-rich zone (CZ)-localizing Nestin-GFP-positive and Nestin-GFP-negative cells proliferate and differentiate into odontoblast-like cells in response to odontoblast depletion. The regenerated odontoblast-like cells played a role in reparative dentin formation. RNA-sequencing analysis revealed that the expression of odontoblast differentiation- and activation-related genes was upregulated in the pulp in response to odontoblast depletion even without damage to dental tissue. In this regenerative process, the expression of type I parathyroid hormone receptor (PTH1R) increased in the odontoblast-depleted pulp, thereby boosting dentin formation. The levels of PTH1R and its downstream mediator, i.e., phosphorylated cyclic AMP response element-binding protein (Ser133) increased in the physically damaged pulp. Collectively, odontoblast death triggered the PTH1R cascade, which may represent a therapeutic target for inducing CZ-mediated dental regeneration.

Administration of the bisphosphonate zoledronic acid during tooth development inhibits tooth eruption and formation and induces dental abnormalities in rats.

Bisphosphonates (BPs) are potent inhibitors of osteoclastic bone resorption and widely used for the treatment of osteoporosis and metastatic bone diseases. Recently, BPs have also been shown to benefit children with primary and secondary osteoporosis, including osteogenesis imperfecta; however, their long-term safety has not been established yet. Clinical and experimental studies have demonstrated that BPs delay or inhibit tooth eruption. The failure of tooth eruption causes several dental abnormalities. In this study, to determine the effects of BPs on tooth formation, the BP zoledronic acid (ZOL) was injected into 7- and 14-day-old rats, and the development of the mandibular teeth was examined. X-ray analysis demonstrated that ZOL inhibited the eruption of both incisors and molars and their formation, especially in the molar roots. Histological examination showed that, in ZOL-treated animals, alveolar bone remained unresorbed around tooth crowns, which injured ameloblasts and enamel matrix, leading to defects of the enamel. Furthermore, haphazard proliferation of odontogenic epithelium and mesenchyme associated with primitive tooth structures, which resembles human odontomas, was induced at the basal end of incisors but not around the molars. Tooth ankylosis to alveolar bone was occasionally observed in molars. These results suggest that administration of BPs during tooth development has the potential to inhibit tooth eruption and formation and to induce several types of dental abnormalities, which may be attributed to the altered osteoclastic activities.