Macrophages are involved in tissue inflammation and repair through cytokine secretion. However, the contribution of macrophages to healing and osteogenesis after tooth extraction remains unclear. Therefore, we investigated the distribution of osteoblastic cells and macrophages in the early healing process after tooth extraction. The maxillary first molars of 6-week-old male mice were extracted. The maxilla was collected 1, 3, and 7 days after extraction. The states of socket healing, localization of osteoblastic markers, and macrophage infiltration were sequentially observed by micro-CT imaging and immunohistochemistry. On day 3 after tooth extraction, α-smooth muscle actin (SMA)-positive cells, osteoprogenitor cells at fracture healing, were observed in the socket. Several α-SMA-positive cells also expressed Runx2, the early osteoblast differentiation marker. The infiltration of F4/80-positive, mature macrophages and CD206-positive, M2-like macrophages was noted in the socket. However, CD169-positive macrophages (Osteomac), which are involved in fracture healing, were not detected in the socket. F4/80-positive and CD206-positive macrophages also showed the localization of transforming growth factor-β (TGF-β), which promotes osteoprogenitor cell proliferation and early differentiation. Phosphorylated Smad3, a downstream mediator of the signal activity of TGF-β, was detected in α-SMA-positive cells. On day 7, the extracted socket contained a large amount of new bone. Tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts were detected on bone surfaces. Our data indicate that M2-like macrophages regulate the proliferation and differentiation of α-SMA-positive cells by secreting TGF-β at the early stage of socket healing, and also suggest the importance of macrophages in healing and bone formation after tooth extraction.

Wnt signaling has been reported to be involved in dentin bridge formation. However, the detailed mechanism has not yet been clarified. We elucidated the localization of canonical Wnt signaling molecules during dentin bridge formation. Pulp of the maxillary first molar in mice was exposed and directly capped with MTA cement. Maxillae were collected on the 1st, 4th, 7th, 14th, and 28th days after treatment. After μCT analysis, immunohistochemistry for Wnt3a, Wnt10a, β-catenin, F4/80, and osterix was performed in paraffin-embedded sections. On the 4th and 7th days after pulp capping, odontoblasts and dental pulp cells expressed Wnt3a, Wnt10a, and β-catenin. On the 14th day, reactionary dentin was formed around the pulp exposure area. Odontoblasts and dental pulp cells express Wnt3a, Wnt10a, and β-catenin. Additionally, F4/80- and Wnt10a-positive macrophages were observed at the center of the dental pulp. When the dentin bridge was formed on the 28th day, reparative odontoblasts expressed Wnt3a, β-catenin and osterix. Wnt ligands derived from odontoblasts and dental pulp cells are important for the activation of odontoblasts and the differentiation of reparative odontoblasts during dentin bridge formation. Macrophage-derived Wnts are also involved in reparative odontoblast differentiation.

Chlorogenic acid (CGA) is a polyphenol that is present in coffee beans, many vegetables, and fruits. Since CGA has been reported to exert antioxidant and anti-inflammatory effects, it is expected to protect against periodontitis. In the present study, we used a ligature-induced experimental periodontitis model and investigated the beneficial effects of CGA against alveolar bone resorption caused by experimental periodontitis. To examine the inhibitory effects of CGA on bone loss, a ligature was wrapped around the maxillary right second molar, and CGA was intraperitoneally injected once a day for 2 weeks. In another experiment to investigate the restorative effects of CGA on bone loss, a ligature was wrapped around the maxillary right second molar for 2 weeks, it was then removed, and CGA was intraperitoneally injected once a day for 2 weeks. At the end of the experiments, the maxillae were removed, and CT images were taken. Alveolar bone loss was measured as the distance from the cement–enamel junction to the alveolar crest. The statistical analysis was performed using GraphPad Prism6 (Dunn’s multiple comparison test). The results revealed that the ratio of the buccal alveolar bone loss (vs. the bone loss on the nonligated side) induced by ligation was significantly decreased by the administration of CGA (5 mg/kg) for 2 weeks. Moreover, the bone loss ratio on the buccal and palatal sides after 2 weeks of ligation was significantly decreased by the 2-week administration of CGA (5 mg/kg). The present results revealed that CGA exerted preventive effects against alveolar bone loss caused by experimental periodontitis.