Abstract As the pulp regeneration for non-vital teeth is one of the ultimate clinical achievements, regenerative endodontic procedures (REPs) have become the most explored treatment modality. In this technique, periodontal tissue is guided from the apical region into the root canal and pulp chamber to promote attachment. It is well established that immature teeth are effective targets for treatment. However, the indications for this treatment have not yet expanded sufficiently to encompass mature teeth with closed apical apex. In the present study, a mouse model of REPs in mature teeth was established, employing the maxillary first molar mesial root. μCT analyses disclosed that the distance from the occlusal surface to the physiological apex of the maxillary first molar mesial root in mice is 2.14 mm ± 0.08 mm, and the distance from the occlusal surface to the periapical alveolar bone is 2.46 mm ± 0.10 mm. Mesial root canal was treated with several sizes of k-files, and 15# k-file was identified as the most suitable k-file for use (P = 0.0007). During the regenerative process, spindle-shaped fibroblast-like cells, fibrous tissue formation, and mineralized tissue formation were identified on days 14 and 28. This study demonstrated that it is feasible to use the maxillary first molar mesial root as a REPs model for mature teeth and provided a detailed protocol and analysis of the healing process.

目的：局所クロマチンの「ゆるみ」によるクロマチンアクセシビリティの上昇は，転写複合体の結合を可能にすることで標的遺伝子発現を促すことから，エピジェネティクスな遺伝子発現調節機構の主体である．そこで本研究では，いまだ明らかになっていないヒト歯髄幹細胞（human dental pulp stem cells：hDPSC）の分化過程における全ゲノム的なクロマチンアクセシビリティの変化をATAC-seq（Assay for Transposase-Accessible Chromatin with high-throughput sequencing）により捉えることを目的とする．  材料および方法：hDPSCを石灰化誘導培地で12日間培養し，培養前後にATAC-seq用サンプルの調製を行った．バイオインフォマティクス解析により，オープンクロマチンピーク抽出，サンプル間比較，コンセンサスDNA結合配列（CDB）の同定，各ピークの近傍遺伝子に対するGene Ontology（GO）解析を行った．  結果：培養0日目と12日目において有意なオープンクロマチンピークをそれぞれ45,493個と45,370個同定した．これらオープンクロマチンピークにはTEAD，RUNX，bZIPなどの転写因子群とCTCF，Borisというインシュレーター（局所クロマチンの区切り壁を形成する因子）のCDBが共通して集積していた．さらにCTCF-CDB近傍遺伝子のGO解析では，培養12日目においてのみ，TEADによって転写制御を受けるHippo signaling pathwayが上位にランクされた．CTCF-CDB近傍のHippo signaling pathwayに属する遺伝子はACTB，ACTG1，AREG，APC，DLG2，DVL1，BMP2，BMPR1B，NF2，PARD6B，SMAD2であり，BMP2やBMPR1Bなど硬組織形成分化に必須の遺伝子座が含まれていた．以上より，hDPSCの分化過程において，オープンクロマチン領域から検出されるCDBの種類に変化は少ないものの，CTCFによる局所クロマチンの3D構造変化が分化指向性遺伝子座の選択的オープンクロマチン化を引き起こすことで分化指向性遺伝子の発現が誘導されることが示唆された．  結論：hDPSCの分化には各クロマチン領域においてインシュレーターが担うエピジェネティクスな遺伝子発現制御機構が存在しており，その統合的制御によりhDPSCの効率的な分化誘導法が確立できる可能性が示された．

<jats:sec><jats:title>Introduction</jats:title><jats:p>Bone sialoprotein (BSP) is a key regulator of mineralized tissue formation. Previously, we generated BSP-KAE knock-in mice (KAEKI mice) by substituting a non-function KAE (lysine-alanine-glutamic acid) for the integrin-binding RGD (arginine-glycine-aspartic acid) sequence and reported a vital role of the BSP-RGD motif in modulating the periodontal ligament (PDL). Specifically, histologically a disorganization of the PDL was noted, resulting in a weakened function of the PDL as measured by dynamic mechanical analysis. Intriguingly, also noted was a weight gain as KAEKI mice aged. While several proteins associated with mineralized tissues are reported to affect energy metabolism, the metabolic role of the BSP-RGD region has yet to be elucidated. Here we focus on defining the role of the BSP-RGD region in metabolic activity.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>Body weight, body composition, and caloric intake were measured in wild type (WT) and KAEKI mice. Energy expenditure was estimated using energy balance technique. Epididymal fat, interscapular fat, and liver were harvested for histological analysis. Systemic metabolic phenotype was assessed by sera analyses, insulin tolerance and glucose tolerance tests.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>The results showed that KAEKI mice developed mild obesity starting from 13 weeks postnatal (wpn). The increase in body weight correlated with an increase in lean mass and visceral adiposity. Histological examination revealed adipocyte hypertrophy in white epididymal fat and interscapular brown fat in KAEKI vs. WT mice at 17 wpn. Metabolic profiling indicated that KAEKI mice had dyslipidemia and hyperleptinemia but no significant changes in glucose metabolism. Energy balance analyses revealed that hyperphagia preceded weight gain in KAEKI mice.</jats:p></jats:sec><jats:sec><jats:title>Conclusion</jats:title><jats:p>These data suggest that the RGD region of BSP affects energy metabolism by regulating food intake, with further studies warranted to uncover the underlying mechanisms.</jats:p></jats:sec>

<jats:p>A better understanding of the role of mineralized tissues and their associated factors in governing whole-body metabolism should be of value toward informing clinical strategies to treat mineralized tissue and metabolic disorders, such as diabetes and obesity. This perspective provides evidence suggesting a role for the arginine-glycine-aspartic acid (RGD) region, a sequence identified in several proteins secreted by bone cells, as well as other cells, in modulating systemic metabolic activity. We focus on (a) two of the SIBLING (<jats:bold>s</jats:bold>mall <jats:bold>i</jats:bold>ntegrin-<jats:bold>b</jats:bold>inding <jats:bold>li</jats:bold>gand, <jats:bold>N</jats:bold>-linked <jats:bold>g</jats:bold>lycoprotein) family genes/proteins, bone sialoprotein (BSP) and osteopontin (OPN), (b) insulin-like growth factor-binding protein-1 &amp;amp; 2 (IGFBP-1, IGFBP-2) and (c) developmental endothelial locus 1 (DEL1) and milk fat globule–EGF factor-8 (MFG-E8). In addition, for our readers to appreciate the mounting evidence that a multitude of bone secreted factors affect the activity of other tissues, we provide a brief overview of other proteins, to include fibroblast growth factor 23 (FGF23), phosphatase orphan 1 (PHOSPHO1), osteocalcin (OCN/BGLAP), tissue non-specific alkaline phosphatase (TNAP) and acidic serine aspartic-rich MEPE-associated motif (ASARM), along with known/suggested functions of these factors in influencing energy metabolism.</jats:p>

<jats:p> Bone sialoprotein (gene: Ibsp; protein: BSP) is a multifunctional extracellular matrix protein present in bone, cementum, and dentin. Accumulating evidence supports BSP as a key regulator of mineralized tissue formation via evolutionarily conserved functional domains, including a C-terminal integrin-binding Arg-Gly-Asp (RGD) domain implicated in extracellular matrix–cell signaling. Ablation of Ibsp in mice ( Ibsp<jats:sup>−/−</jats:sup>) results in impaired bone growth and mineralization and defective osteoclastogenesis, with effects in the craniofacial region including reduced acellular cementum formation, detachment of the periodontal ligament (PDL), alveolar bone hypomineralization, and severe periodontal breakdown. We hypothesized that BSP-RGD plays an important role in cementum and alveolar bone formation and mineralization, as well as periodontal function. This hypothesis was tested by replacing the RGD motif with a nonfunctional Lys-Ala-Glu (KAE) sequence in ( Ibsp<jats:sup>KAE/KAE</jats:sup>) mice and OCCM.30 murine ( Ibsp<jats:sup>KAE</jats:sup>) cementoblasts. The RGD domain was not critical for acellular or cellular cementum formation in Ibsp<jats:sup>KAE/KAE</jats:sup> mice. However, PDL volume and thickness were increased, and significantly more tartrate-resistant acid phosphatase–positive osteoclasts were found on alveolar bone surfaces of Ibsp<jats:sup>KAE/KAE</jats:sup> mice versus wild type mice. PDL organization was disrupted as indicated by picrosirius red stain, second harmonic generation imaging, dynamic mechanical analysis, and decreased asporin proteoglycan localization. In vitro studies implicated RGD functions in cell migration, adhesion, and mineralization, and this was confirmed by an ossicle implant model where cells lacking BSP-RGD showed substantial defects as compared with controls. In total, the BSP-RGD domain is implicated in periodontal development, though the scale and scope of changes indicated by in vitro studies indicate that other factors may partially compensate for and reduce the phenotypic severity of mice lacking BSP-RGD in vivo. </jats:p>