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.

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.

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.