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.

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.

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.

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.

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.

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.

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.

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.

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.

Localization of RELM-ß/FIZZ2 Is Associated with Cementum Formation.

Resistin-like molecule-ß/found in inflammatory zone 2 (RELM-ß/FIZZ2) is a cysteine-rich secretory protein that is localized in the epithelium of the gastrointestinal tract and lung alveoli. Previous reports have suggested that this protein regulates glucose metabolism and inflammation. In the present study, to analyze the involvement of RELM-ß/FIZZ2 in tooth development, we immunohistochemically examined the localization of RELM-ß/FIZZ2 in tooth germs of embryonic days (E) 15-20 and postnatal days (P) 7-42 rats. RELM-ß/FIZZ2 was hardly detected in the tooth germ at the bud (E15) stage. However, at the cap (E17) and bell (E20) stages, this protein was detectable in the inner enamel epithelium; whereas cells in the other parts of the enamel organ including the outer enamel epithelium and stellate reticulum did not show the immunoreactivity. During the root formation stage (P14-28), cells in Hertwig's epithelial root sheath (HERS) localized RELM-ß/FIZZ2. Intense immunoreactivity was also seen in the matrix of the root dentin facing the HERS and the dental follicle. This reactivity was not present on the more upwardly located dentin surface. In contrast, cementum matrix positive for osteopontin and bone sialoprotein was observed on the dentin instead of immunoreactivity for RELM-ß/FIZZ2. Osterix-positive cells, indicating cementoblast progenitors, were also detected in the dental follicle near the matrix positive for RELM-ß/FIZZ2. These results suggest that RELM-ß/FIZZ2 secreted by the inner enamel epithelium was mainly localized in the matrix at the surface of the apical root dentin and might be involved in cementogenesis. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 300:1865-1874, 2017. © 2017 Wiley Periodicals, Inc.

VDR in Osteoblast-Lineage Cells Primarily Mediates Vitamin D Treatment-Induced Increase in Bone Mass by Suppressing Bone Resorption.

Long-term treatment with active vitamin D [1α,25(OH)2 D3 ] and its derivatives is effective for increasing bone mass in patients with primary and secondary osteoporosis. Derivatives of 1α,25(OH)2 D3 , including eldecalcitol (ELD), exert their actions through the vitamin D receptor (VDR). ELD is more resistant to metabolic degradation than 1α,25(OH)2 D3 . It is reported that ELD treatment causes a net increase in bone mass by suppressing bone resorption rather than by increasing bone formation in animals and humans. VDR in bone and extraskeletal tissues regulates bone mass and secretion of osteotropic hormones. Therefore, it is unclear what types of cells expressing VDR preferentially regulate the vitamin D-induced increase in bone mass. Here, we examined the effects of 4-week treatment with ELD (50 ng/kg/day) on bone using osteoblast lineage-specific VDR conditional knockout (Ob-VDR-cKO) and osteoclast-specific VDR cKO (Ocl-VDR-cKO) male mice aged 10 weeks. Immunohistochemically, VDR in bone was detected preferentially in osteoblasts and osteocytes. Ob-VDR-cKO mice showed normal bone phenotypes, despite no appreciable immunostaining of VDR in bone. Ob-VDR-cKO mice failed to increase bone mass in response to ELD treatment. Ocl-VDR-cKO mice also exhibited normal bone phenotypes, but normally responded to ELD. ELD-induced FGF23 production in bone was regulated by VDR in osteoblast-lineage cells. These findings suggest that the vitamin D treatment-induced increase in bone mass is mediated by suppressing bone resorption through VDR in osteoblast-lineage cells. © 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.

Correlation between Fibrillin-1 Degradation and mRNA Downregulation and Myofibroblast Differentiation in Cultured Human Dental Pulp Tissue.

Myofibroblasts and extracellular matrix are important components in wound healing. Alpha-smooth muscle actin (α-SMA) is a marker of myofibroblasts. Fibrillin-1 is a major constituent of microfibrils and an extracellular-regulator of TGF-ß1, an important cytokine in the transdifferentiation of resident fibroblasts into myofibroblasts. To study the correlation between changes in fibrillin-1 expression and myofibroblast differentiation, we examined alterations in fibrillin-1 and α-SMA expression in organotypic cultures of dental pulp in vitro. Extracted healthy human teeth were cut to 1-mm-thick slices and cultured for 7 days. In intact dental pulp, fibrillin-1 was broadly distributed, and α-SMA was observed in pericytes and vascular smooth muscle cells. After 7 days of culture, immunostaining for fibrillin-1 became faint concomitant with a downregulation in its mRNA levels. Furthermore, fibroblasts, odontoblasts and Schwann cells were immunoreactive for α-SMA with a significant increase in α-SMA mRNA expression. Double immunofluorescence staining was positive for pSmad2/3, central mediators of TGF-ß signaling, and α-SMA. The administration of inhibitors for extracellular matrix proteases recovered fibrillin-1 immunostaining; moreover, fibroblasts lost their immunoreactivity for α-SMA along with a downregulation in α-SMA mRNA. These findings suggest that the expression of α-SMA is TGF-ß1 dependent, and fibrillin-1 degradation and downregulation might be implicated in the differentiation of myofibroblasts in dental pulp wound healing.

Enhanced bone-forming activity of side population cells in the periodontal ligament.

Regeneration of alveolar bone is critical for the successful treatment of periodontal diseases. The periodontal ligament (PDL) has been widely investigated as a source of cells for the regeneration of periodontal tissues. In the present study where we attempted to develop an effective strategy for alveolar bone regeneration, we examined the osteogenic potential of side population (SP) cells, a stem cell-containing population that has been shown to be highly abundant in several kinds of tissues, in PDL cells. Isolated SP cells from the rat PDL exhibited a superior ability to differentiate into osteoblastic cells compared with non-SP (NSP) and unsorted PDL cells in vitro. The mRNA expressions of osteoblast markers and bone morphogenetic protein (BMP) 2 were significantly upregulated in SP cells and were further increased by osteogenic induction. To examine the bone-forming activity of SP cells in vivo, PDL SP cells isolated from green fluorescent protein (GFP)-transgenic rats were transplanted with hydroxyapatite (HA) disks into wild-type animals. SP cells exhibited a high ability to induce the mineralized matrix compared with NSP and unsorted PDL cells. At 12 weeks after the implantation, some of the pores in the HA disks with SP cells were filled with mineralized matrices, which were positive for bone matrix proteins, such as osteopontin, bone sialoprotein, and osteocalcin. Furthermore, osteoblast- and osteocyte-like cells on and in the bone-like mineralized matrices were GFP positive, suggesting that the matrices were directly formed by the transplanted cells. These results suggest that PDL SP cells possess enhanced osteogenic potential and could be a potential source for cell-based regenerative therapy for alveolar bone.