Periodontal ligament (PDL) possesses a stem/progenitor population to maintain the homeostasis of periodontal tissue. However, transcription factors that regulate this population have not yet been identified. Thus, we aimed to identify a molecule related to the osteogenic differentiation of PDL progenitors using a single cell-based strategy in this study. We first devised a new protocol to isolate PDL cells from the surface of adult murine molars and established 35 new single cell-derived clones from the PDL explant. Among these clones, six clones with high (high clones, n = 3) and low (low clones, n = 3) osteogenic potential were selected. Despite a clear difference in the osteogenic potential of these clones, no significant differences in their cell morphology, progenitor cell marker expression, alkaline phosphatase activity, proliferation rate, and differentiation-related gene and protein expression were observed. RNA-seq analysis of these clones revealed that Z-DNA binding protein-1 (Zbp1) was significantly expressed in the high osteogenic clones, indicating that Zbp1 could be a possible marker and regulator of the osteogenic differentiation of PDL progenitor cells. Zbp1-positive cells were distributed sparsely throughout the PDL. In vitro Zbp1 expression in the PDL clones remained at a high level during osteogenic differentiation. The CRISPR/Cas9 mediated Zbp1 knockout in the high clones resulted in a delay in cell differentiation. On the other hand, Zbp1 overexpression in the low clones promoted cell differentiation. These findings suggested that Zbp1 marked the PDL progenitors with high osteogenic potential and promoted their osteogenic differentiation. Clarifying the mechanism of differentiation of PDL cells by Zbp1 and other factors in future studies will facilitate a better understanding of periodontal tissue homeostasis and repair, possibly leading to the development of novel therapeutic measures.

Periodontal tissue supports teeth in the alveolar bone socket via fibrous attachment of the periodontal ligament (PDL). The PDL contains periodontal fibroblasts and stem/progenitor cells, collectively known as PDL cells (PDLCs), on top of osteoblasts and cementoblasts on the surface of alveolar bone and cementum, respectively. However, the characteristics and lineage hierarchy of each cell type remain poorly defined. This study identified periodontal ligament associated protein-1 (Plap-1) as a PDL-specific extracellular matrix protein. We generated knock-in mice expressing CreERT2 and GFP specifically in Plap-1-positive PDLCs. Genetic lineage tracing confirmed the long-standing hypothesis that PDLCs differentiate into osteoblasts and cementoblasts. A PDL single-cell atlas defined cementoblasts and osteoblasts as Plap-1−Ibsp+Sparcl1+ and Plap-1−Ibsp+Col11a2+, respectively. Other populations, such as Nes+ mural cells, S100B+ Schwann cells, and other non-stromal cells, were also identified. RNA velocity analysis suggested that a Plap-1highLy6a+ cell population was the source of PDLCs. Lineage tracing of Plap-1+ PDLCs during periodontal injury showed periodontal tissue regeneration by PDLCs. Our study defines diverse cell populations in PDL and clarifies the role of PDLCs in periodontal tissue homeostasis and repair.

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Hard tissues, including the bones and teeth, are a fundamental part of the body, and their formation and homeostasis are critically regulated by matrix vesicle–mediated mineralization. Matrix vesicles have been studied for 50 y since they were first observed using electron microscopy. However, research progress has been hampered by various technical barriers. Recently, there have been great advancements in our understanding of the intracellular biosynthesis of matrix vesicles. Mitochondria and lysosomes are now considered key players in matrix vesicle formation. The involvement of mitophagy, mitochondrial-derived vesicles, and mitochondria–lysosome interaction have been suggested as potential detailed mechanisms of the intracellular pathway of matrix vesicles. Their main secretion pathway may be exocytosis, in addition to the traditionally understood mechanism of budding from the outer plasma membrane. This basic knowledge of matrix vesicles should be strengthened by novel nano-level microscopic technologies, together with basic cell biologies, such as autophagy and interorganelle interactions. In the field of tissue regeneration, extracellular vesicles such as exosomes are gaining interest as promising tools in cell-free bone and periodontal regenerative therapy. Matrix vesicles, which are recognized as a special type of extracellular vesicles, could be another potential alternative. In this review, we outline the recent significant progress in the process of matrix vesicle–mediated mineralization and the potential clinical applications of matrix vesicles for tissue regeneration.

Objective To investigate the mutual regulation of hypoxia‐inducible factor (HIF)‐1α activity and periodontal ligament‐associated protein‐1 (PLAP‐1) expression in human periodontal ligament cells (HPDLs). Background Cellular responses to hypoxia regulate various biological events (e.g., inflammation and tissue regeneration) through activation of HIF‐1α. PLAP‐1, an extracellular matrix protein preferentially expressed in the periodontal ligament, plays important roles in the functions of HPDLs. Although PLAP‐1 expression has been demonstrated in hypoxic regions, the involvement of PLAP‐1 in responses to hypoxia has not been revealed. Methods HPDLs were cultured under normoxic (20% O2) or hypoxic (1% O2) conditions with or without deferoxamine mesylate (chemical hypoxia inducer) or chetomin (HIF signaling inhibitor). Expression levels of PLAP‐1 and HIF‐1α were examined by real‐time reverse transcription‐polymerase chain reaction and western blot analysis. Luciferase reporter assays of HIF‐1α activity were performed using 293T cells stably transfected with a hypoxia response element (HRE)‐containing luciferase vector in the presence or absence of recombinant PLAP‐1 or PLAP‐1 gene transfection. Results Cultivation under hypoxic conditions elevated the gene and protein expression levels of PLAP‐1 in HPDLs. Deferoxamine mesylate treatment also enhanced PLAP‐1 expression in HPDLs. Hypoxia‐induced PLAP‐1 expression was significantly suppressed in the presence of chetomin. PLAP‐1‐suppressed HPDLs showed increased HIF‐1α accumulation in the nucleus during culture under hypoxic conditions, but not in the presence of recombinant PLAP‐1. In the presence of recombinant PLAP‐1, hypoxia‐induced HRE activity of 293T cells was significantly suppressed in a dose‐dependent manner. Transfection of the PLAP‐1 gene resulted in a significant reduction of HRE activity during culture under hypoxic conditions. Conclusion PLAP‐1 expression is upregulated under hypoxic conditions through HIF‐1α activation. Moreover, hypoxia‐induced PLAP‐1 expression regulates HIF‐1α signaling.