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.

Purpose: An increase in chromatin accessibility arises from the stretched chromatin structure, which enables the association of transcription complexes with local genomic DNA, and thus induces target mRNA transcription. This is one of the main molecular frameworks for epigenetic regulation of gene expression. In this study, the changes in whole-genomic chromatin accessibility that occur during induced differentiation of human dental pulp stem cells (hDPSC) were analyzed by assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq).  Methods: hDPSC were cultured in a mineralization induction medium. ATAC-seq samples were prepared before and after the 12-day culture. Bioinformatics analyses were conducted, involving open chromatin peak extraction, sample comparison, consensus DNA binding (CDB) sequence identification, and gene ontology (GO) analysis of neighboring genes for each peak.  Results: The bioinformatics analyses identified 45,493 and 45,370 open chromatin peaks at day 0 and day 12, respectively. The CDB sequences of transcription factors, including TEADs, bZIPs, and RUNXs, and insulators, including CTCF and BORIS, were commonly enriched in these peaks. Furthermore, GO analysis of neighboring genes of CTCF-CDB revealed an accumulation of the genes associated with the Hippo signaling pathway, in which TEADs act as the essential transcription factors, at day 12 but not at day 0. Additionally, the gene loci of the odonto/osteogenic genes, such as BMP2 and BMPR1B, were identified as accessible chromatin regions controlled by CTCF-CDB at day 12 but not at day 0. From these results, the types of CDB in the stretched chromatin region were not drastically changed before and after hDPSC differentiation. However, because CTCF participates in the dynamic genomic organization to regulate local chromatin accessibility and restrict the territory of distal enhancers and suppressors, CTCF aids TEADs in finding and associating with odonto/osteogenic gene loci, inducing odonto/osteogenic gene expression during differentiation.  Conclusion: Bioinformatics analysis indicated that local epigenetic alteration mediated by the insulators may play pivotal roles in hDPSC differentiation, and therefore, chromatin accessibility modulation may have therapeutic potential for inducing odontogenic differentiation of hDPSC.

<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>