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The aim of this study is to clarify the biological functions of decorin (DCN) in the healing and regeneration of wounded periodontal tissue. We investigated the expression pattern of DCN during the healing of wounded periodontal tissue in rats by immunohistochemistry and the effects of DCN on the osteoblastic differentiation of human periodontal ligament (PDL) stem cells (HPDLSCs) and preosteoblasts by Alizarin red S staining, quantitative reverse transcription-polymerase chain reactions, and western blotting. The expression of DCN was increased around the wounded PDL tissue on day 5 after surgery compared with the nonwounded PDL tissue, whereas its expression was not changed in the osteoblastic layer around the wounded alveolar bone. Furthermore, DCN promoted the osteoblastic differentiation of HPDLSCs, but it did not affect the osteoblastic differentiation of preosteoblasts. ERK1/2 phosphorylation was upregulated during the DCN-induced osteoblastic differentiation of HPDLSCs. DCN did not affect proliferation, migration, or the PDL-related gene expression of HPDLSCs. In conclusion, this study demonstrates that DCN has a role in the healing of wounded periodontal tissue. Furthermore, DCN secreted from PDL cells may contribute to bone healing by upregulating osteoblastic differentiation through ERK1/2 signaling in HPDLSCs, indicating a therapeutic effect of DCN in periodontal tissue regeneration.

In cases in which dental pulp tissue is accidentally exposed, direct pulp capping is often performed to induce reparative dentin formation. Although macrophages are essential for the inflammatory response and tissue repair, the emergence pattern and the role of macrophages in dental pulp tissue have not been clarified. Here, we investigated the emergence of M1/M2 macrophages in dental pulp tissue after a direct pulp capping and the effects of M2 macrophages on odontoblastic differentiation of the dental pulp stem cell (DPSC) clones. The emergence of macrophages in dental pulp tissue was investigated using a rat direct pulp capping model. Alizarin Red S staining and quantitative RT-PCR was performed to examine the effect of M2 macrophages on the mineralization and odontoblastic differentiation of DPSC clones. Immunohistochemical staining revealed that M1 macrophages were detected in dental pulp tissue after treatment and increased in number at three days after treatment. However, M2 macrophages gradually increased in number in dental pulp tissue after treatment, with the highest level recorded at seven days post-operation. Additionally, conditioned medium from M2 macrophages induced odontoblast-like differentiation of DPSC clones. These results suggest that macrophages play a role in the inflammatory response and reparative dentin formation after dental pulp exposure.

Periodontal ligament stem cells (PDLSCs) play central roles in periodontal ligament (PDL) tissue homeostasis, repair, and regeneration. Previously, we established a protocol to differentiate human-induced pluripotent stem cell-derived neural crest-like cells (iNCs) into PDLSC-like cells (iPDLSCs) using human PDL cell-derived extracellular matrix (ECM). However, it remained unclear what factors principally regulate the differentiation of iNCs into iPDLSCs. In this study, we aimed to identify the transcription factor regulating production of human PDL cell-derived ECM, which is responsible for the generation of iPDLSCs. We cultured iNCs on ECMs of two human PDL cell lines (HPDLC-3S and HPDLC-3U) and of human dermal fibroblasts (HDF). iNCs cultured on HPDLC-3U demonstrated higher iPDLSC-associated gene expression and mesenchymal differentiation capacity than cells cultured on HDF or HPDLC-3S. The transcription factor PAX9 was highly expressed in HPDLC-3U compared with HDF and HPDLC-3S. iNCs cultured on siPAX9-transfected HPDLC-3U displayed downregulation of iPDLSC-associated marker expression and adipocytic differentiation capacity relative to controls. Our findings suggest that PAX9 is one of the transcription factors regulating ECM production in human PDL cells, which is responsible for the differentiation of iNCs into iPDLSCs.

In the case of dental pulp exposure, direct pulp capping is often performed to preserve vital dental pulp tissue. Numerous studies regarding the development of direct pulp-capping materials have been conducted, but materials with an appropriate sealing ability, which induce dense reparative dentin formation, have not been developed. Although nano hydroxyapatite (naHAp) is a bone-filling material with bioactivity and biocompatibility, the inductive effects of naHAp on reparative dentin formation remain unclear. In the present study, the effects of dental adhesive material 4-methacryloxyethyl trimellitate anhydride/methyl methacrylate tri-n-butylborane [4-META/MMA-TBB or Super-bond (SB)], which included 10%, 30%, and 50% naHAp (naHAp/SB) on odontoblastic differentiation of dental pulp stem cells (DPSCs) and reparative dentin formation were investigated. Scanning electron microscopy (SEM) and energy dispersive X-ray spectrometer analysis were performed to verify the existence of naHAp particles on the surface of naHAp/SB discs. The tensile adhesive strength of naHAp/SB was measured using a universal testing machine. As a result, 10% naHAp/SB and 30% naHAp/SB showed almost the same tensile adhesive strength as SB but 50% naHAp/SB showed significantly lower than the other experimental group. WST-1 proliferation assay and SEM analysis revealed that naHAp/SB did not affect the proliferation of DPSCs. Calcium release assay, quantitative RT-PCR, and western blotting analysis demonstrated that naHAp/SB did not release calcium ion but 30% naHAp/SB increased the expression of calcium-sensing receptor (CaSR) in DPSCs. Additionally, quantitative RT-PCR, western blotting analysis, Alizarin Red S- and von Kossa staining revealed that 30% naHAp/SB induced odontoblastic differentiation of DPSCs, which was inhibited by a MEK/ERK inhibitor and CaSR antagonist. Furthermore, 30% naHAp/SB promoted dense reparative dentin formation in an experimentally-formed rat dental pulp exposure model. These findings suggest that 30% naHAp/SB can be used as an ideal direct pulp capping material., (Copyright © 2021 Elsevier B.V. All rights reserved.)