Takenobu Ishii

Soluble RANKL Cleaved from Activated Lymphocytes by TNF-α–Converting Enzyme Contributes to Osteoclastogenesis in Periodontitis

Host immune responses play a key role in promoting bone resorption in periodontitis via receptor activator of NF-κB ligand (RANKL)–dependent osteoclastogenesis. Both membrane-bound RANKL (mRANKL) expressed on lymphocytes and soluble RANKL (sRANKL) are found in periodontal lesions. However, the underlying mechanism and cellular source of sRANKL release and its biological role in periodontitis are unclear. TNF-α–converting enzyme (TACE) is reported to cleave the following: 1) precursor TNF-α with release of mature, soluble TNF-α and 2) mRANKL with release of sRANKL. Both soluble TNF-α and sRANKL are found in the periodontitis lesion, leading to the hypothesis that TACE expressed on lymphocytes is engaged in RANKL shedding and that the resulting sRANKL induces osteoclastogenesis. In the current study, upon stimulating PBLs with mitogens in vitro, RANKL expression, sRANKL secretion, and TACE expression were all upregulated. Among the four putative mRANKL sheddases examined in neutralization assays, TACE was the only functional sheddase able to cleave mRANKL expressed on PBL. Moreover, PBL culture supernatant stimulated with mitogens in the presence of anti-TACE Ab or anti-RANKL Ab showed a marked reduction of osteoclastogenesis from osteoclast precursors, indicating that TACE-mediated sRANKL may possess sufficient osteoclastogenic activity. According to double-color confocal microscopy, B cells expressed a more pronounced level of RANKL and TACE expression than T cells or monocytes in periodontally diseased gingiva. Conditioned medium of patients’ gingival lymphocyte culture increased in vitro osteoclastogenic activity, which was suppressed by the addition of anti-TACE Ab and anti-RANKL Ab. Therefore, TACE-mediated cleavage of sRANKL from activated lymphocytes, especially B cells, can promote osteoclastogenesis in periodontitis.

Macrophage Migration Inhibitory Factor (MIF) Supports Homing of Osteoclast Precursors to Peripheral Osteolytic Lesions

By binding to its chemokine receptor CXCR4 on osteoclast precursor cells (OCPs), it is well known that stromal cell‐derived factor‐1 (SDF‐1) promotes the chemotactic recruitment of circulating OCPs to the homeostatic bone remodeling site. However, the engagement of circulating OCPs in pathogenic bone resorption remains to be elucidated. The present study investigated a possible chemoattractant role of macrophage migration inhibitory factor (MIF), another ligand for C‐X‐C chemokine receptor type 4 (CXCR4), in the recruitment of circulating OCPs to the bone lytic lesion. To accomplish this, we used Csf1r‐eGFP‐knock‐in (KI) mice to establish an animal model of polymethylmethacrylate (PMMA) particle‐induced calvarial osteolysis. In the circulating Csf1r‐eGFP+ cells of healthy Csf1r‐eGFP‐KI mice, Csf1r+/CD11b+ cells showed a greater degree of RANKL‐induced osteoclastogenesis compared to a subset of Csf1r+/RANK+ cells in vitro. Therefore, Csf1r‐eGFP+/CD11b+ cells were targeted as functionally relevant OCPs in the present study. Although expression of the two cognate receptors for MIF, CXCR2 and CXCR4, was elevated on Csf1r+/CD11b+ cells, transmigration of OCPs toward recombinant MIF in vitro was facilitated by ligation with CXCR4, but not CXCR2. Meanwhile, the level of PMMA‐induced bone resorption in calvaria was markedly greater in wild‐type (WT) mice compared to that detected in MIF‐knockout (KO) mice. Interestingly, in contrast to the elevated MIF, diminished SDF‐1 was detected in a particle‐induced bone lytic lesion of WT mice in conjunction with an increased number of infiltrating CXCR4+ OCPs. However, such diminished SDF‐1 was not found in the PMMA‐injected calvaria of MIF‐KO mice. Furthermore, stimulation of osteoblasts with MIF in vitro suppressed their production of SDF‐1, suggesting that MIF can downmodulate SDF‐1 production in bone tissue. Systemically administered anti‐MIF neutralizing monoclonal antibody (mAb) inhibited the homing of CXCR4+ OCPs, as well as bone resorption, in the PMMA‐injected calvaria, while increasing locally produced SDF‐1. Collectively, these data suggest that locally produced MIF in the inflammatory bone lytic site is engaged in the chemoattraction of circulating CXCR4+ OCPs.

Influence of extraoral lateral force loading on the mandible in the mandibular development of growing rats

INTRODUCTION The objective of this study was to elucidate the influence of extraoral lateral force loading of the mandible on mandibular development. METHODS Thirty growing Wistar rats were divided into 3 groups: control, sham, and experimental. To determine longitudinal developmental changes, each animal was placed under anesthesia and immobilized in a custom-built body retainer to allow microcomputed tomography to be performed before and after the experiment. In the experimental group, a fixing device of aluminum was fitted to the zygomatic arch. Lateral force was then applied to the mandible with an open coil for 2 weeks. Stereoscopic images were constructed from 3-dimensional microcomputed tomography images. Absolute lengths and perpendicular heights from the baseline of the lower border of the mandible were measured. The images were superimposed at the baseline planes in each animal. After the final observation, the rats were killed, and the bilateral condyles excised. The condyles were decalcified by the standard method, stained with hematoxylin and eosin and toluidine blue, and observed under a microscope. RESULTS Lateral force induced the mandible to shift toward the nonloaded side; absolute mandibular length at menton-condylion on the side where the load was applied was greater than that in the control group. No difference from the control group was noted on the nonloaded side, and there were no significant differences in perpendicular heights. Lateral loading on the mandible resulted in histopathologic changes: (1) on the side where the load was applied, the cartilaginous zone hypertrophied in the highest margin of the condylar head, the erosive zone expanded, and the width of the mandibular neck decreased; (2) the chondrocyte layer shifted to the medial side on the nonloaded side, and cartilaginous ossification occurred in the lateral direction immediately below the chondrocyte layer, which deformed the mandibular neck toward the medial side and caused asymmetric development of the mandible. CONCLUSIONS These results demonstrate that extraoral lateral force loading during the growth stage causes asymmetrical mandibular development.

DC-STAMP Is an Osteoclast Fusogen Engaged in Periodontal Bone Resorption

Dendritic cell-specific transmembrane protein (DC-STAMP) plays a key role in the induction of osteoclast (OC) cell fusion, as well as DC-mediated immune regulation. While DC-STAMP gene expression is upregulated in the gingival tissue with periodontitis, its pathophysiological roles in periodontitis remain unclear. To evaluate the effects of DC-STAMP in periodontitis, anti-DC-STAMP–monoclonal antibody (mAb) was tested in a mouse model of ligature-induced periodontitis (n = 6–7/group) where Pasteurella pneumotropica (Pp)-reactive immune response activated T cells to produce receptor activator of nuclear factor kappa-B ligand (RANKL), which, in turn, promotes the periodontal bone loss via upregulation of osteoclastogenesis. DC-STAMP was expressed on the cell surface of mature multinuclear OCs, as well as immature mononuclear OCs, in primary cultures of RANKL-stimulated bone marrow cells. Anti-DC-STAMP-mAb suppressed the emergence of large, but not small, multinuclear OCs, suggesting that DC-STAMP is engaged in the late stage of cell fusion. Anti-DC-STAMP-mAb also inhibited pit formation caused by RANKL-stimulated bone marrow cells. Attachment of ligature to a second maxillary molar induced DC-STAMP messenger RNA and protein, along with elevated tartrate-resistant acid phosphatase–positive (TRAP+) OCs and alveolar bone loss. As we expected, systemic administration of anti-DC-STAMP-mAb downregulated the ligature-induced alveolar bone loss. Importantly, local injection of anti-DC-STAMP-mAb also suppressed alveolar bone loss and reduced the total number of multinucleated TRAP+ cells in mice that received ligature attachment. Attachment of ligature induced significantly elevated tumor necrosis factor–α, interleukin-1β, and RANKL in the gingival tissue compared with the control site without ligature (P < 0.05), which was unaffected by local injection with either anti-DC-STAMP-mAb or control-mAb. Neither in vivo anti-Pp IgG antibody nor in vitro anti-Pp T-cell response and resultant production of RANKL was affected by anti-DC-STAMP-mAb. This study illustrated the roles of DC-STAMP in promoting local OC cell fusion without affecting adaptive immune responses to oral bacteria. Therefore, it is plausible that a novel therapeutic regimen targeting DC-STAMP could suppress periodontal bone loss.

Influence of experimental hemi-occlusion on mandibular morphology and internal structure in growing rabbit

Abstract We induced experimental hemi-occlusion in growing rabbits to investigate the effect of masticatory laterality on the morphology and internal structure of the mandible. Three-dimensional morphometric data from micro-CT were used to compare changes in mandibular morphology and internal structure. Rabbits in the experimental group were placed under general anesthesia and their left maxillary and mandibular molars cut as far as the cervical area using a rabbit molar cutter. Thereafter, the teeth were cut once every 2 weeks. Rabbits were kept until they were 17 weeks old. For morphometric analysis of the mandible, we measured mandibular body length, mandibular length, mandibular ramus height and gonial angle. To analyze cancellous bone structure, we measured total volume, cancellous bone volume, cancellous bone volume density, trabecular thickness, trabecular number, trabecular separation, and trabecular spacing. Mandibular ramus height and gonial angle were significantly greater on the occlusal side in the experimental group than on the non-occlusal side. Cancellous bone volume density, trabecular thickness and trabecular number were significantly greater on the occlusal side in the experimental group than on the non-occlusal side, whereas trabecular separation and trabecular spacing were significantly lower on the occlusal side. The results indicate that differences in mechanical stimuli during the growth phase resulting from masticatory dysfunction or parafunction affect the morphology and internal structure of the mandible.

Effect of Platelet-Rich Plasma on Bone Grafting of Alveolar Clefts

Abstract The purpose of this study was to evaluate the effectiveness of platelet-rich plasma (PRP) on the absorption of the bone graft in the alveolar cleft. Twenty-nine patients with alveolar clefts in unilateral cleft lip were examined; 6 were the control group and received iliac cancellous bone and marrow grafts without PRP, while the remaining 23 comprised the PRP group and received grafts with PRP. Quantitative evaluation of remaining bone was made by the computer-aided engineering with multidetector row computed tomography at 1 month and 1 year after surgery. Satisfactory bone bridging formation was observed in all patients. Two patients in control group and 1 patient in PRP group developed wound dehiscence with minor bone exposure. One year postoperatively, the canine was exposed and orthodontically guided into an ideal arch relation in all patients. The mean resorption ratio was 49.9 ± 17.2% and 44.9 ± 14.4% with no significant difference (P = 0.60). In conclusion, there is currently no evidence to suggest that autologous PRP is of value for effect on the bone resorption for alveolar bone graft.

Role of Solid Model Simulation Surgery for Hemifacial Microsomia

Distraction osteogenesis is now a standard procedure for hemifacial microsomia, and various methods have been described. However, it is sometimes difficult to obtain the horizontal occlusal plane and facial symmetry. This brief communication describes and discusses the usefulness of solid model simulation surgery for hemifacial microsomia.

How to revise and utilise the cephalogram for craniofacial dysostosis—Modification of the porion and the McNamara line

McNamara line analysis is often utilised in orthognathic surgery and is particularly useful in that it can easily express the extent of maxillo-mandibular deformity quantitatively. However, in craniofacial dysostosis (CFD), the location of the porion (PR) is sometimes displaced inferiorly. The McNamara line depends on the Frankfurt horizontal plane (FH plane), and hence, its deviation is often extremely distorted in CFD. The objective of this study was to enable more correct reflection of deformities of CFD patients on lateral cephalogram analysis. The lateral cephalograms of 152 Asian individuals were involved. angle basion(BA).orbitale(Or).PR, angle BA. nasion(NA).PR, and the distance from BA to PR on the BA-NA plane were measured and each standard range was established. Using these ranges, modified PR (mPR) is supposed to lie within a fairly small area. Second, we utilised mPR in the practice of McNamara line analysis in CFD patients. In both cases in the present study, it was confirmed that modified McNamara line analysis can express the deformities more precisely than conventinal analysis, as far as the maxillo-mandibular relationship is concerned. However, particularly in Case 2, both the cranio-maxillary and cranio-mandibular relationships were not entirely consistent with the lateral profile appearance even on modified McNamara line analysis.

OC-STAMP promotes osteoclast fusion for pathogenic bone resorption in periodontitis via up-regulation of permissive fusogen CD9

Cell fusion‐mediated formation of multinuclear osteoclasts (OCs) plays a key role in bone resorption. It is reported that 2 unique OC‐specific fusogens [i.e., OC‐stimulatory transmembrane protein (OC‐STAMP) and dendritic cell–specific transmembrane protein (DC‐STAMP)], and permissive fusogen CD9, are involved in OC fusion. In contrast to DC‐STAMP‐knockout (KO) mice, which show the osteopetrotic phenotype, OC‐STAMP‐KO mice show no difference in systemic bone mineral density. Nonetheless, according to the ligature‐induced periodontitis model, significantly lower level of bone resorption was found in OC‐STAMP‐KO mice compared to WT mice. Anti‐OC‐STAMP‐neutralizing mAb down‐modulated in vitro: 1) the emergence of large multinuclear tartrate‐ resistant acid phosphatase‐positive cells, 2) pit formation, and 3) mRNA and protein expression of CD9, but not DC‐STAMP, in receptor activator of NF‐κB ligand (RANKL)‐stimulated OC precursor cells (OCps). While anti–DC‐STAMP‐mAb also down‐regulated RANKL‐induced osteoclastogenesis in vitro, it had no effect on CD9 expression. In our mouse model, systemic administration of anti–OC‐STAMP‐mAb suppressed the expression of CD9 mRNA, but not DC‐STAMP mRNA, in periodontal tissue, along with diminished alveolar bone loss and reduced emergence of CD9+ OCps and tartrate‐resistant acid phosphatase‐positive multinuclear OCs. The present study demonstrated that OC‐STAMP partners CD9 to promote periodontal bone destruction by up‐regulation of fusion during osteoclastogenesis, suggesting that anti–OC‐STAMP‐mAb may lead to the development of a novel therapeutic regimen for periodontitis.—Ishii, T., Ruiz‐Torruella, M., Ikeda, A., Shindo, S., Movila, A., Mawardi, H., Albassam, A., Kayal, R. A., Al‐Dharrab, A. A., Egashira, K., Wisitrasameewong, W., Yamamoto, K., Mira, A. I., Sueishi, K., Han, X., Taubman, M. A., Miyamoto, T., Kawai, T. OC‐STAMP promotes osteoclast fusion for pathogenic bone resorption in periodontitis via up‐regulation of permissive fusogen CD9. FASEB J. 32, 4016–4030 (2018). www.fasebj.org

Difference in Bilateral Timing of Eruption of Permanent Teeth

The aim of this study was to obtain basic data that might serve as criteria in the diagnosis of delayed eruption of the permanent teeth. The synchronicity of the eruption of corresponding contralateral teeth was determined. Data were obtained on both the deciduous and permanent dentition based on records made every 2 months at the Department of Pediatric Dentistry of Tokyo Dental College. These data were then used to investigate bilateral differences in the timing of eruption of the permanent teeth. Over 80% of incisors, first molars, mandibular canines, and maxillary first premolars erupted within 4 months of their contralateral counterparts, while this occurred in 75% of maxillary canines, mandibular first premolars, and mandibular second molars, 70% of maxillary and mandibular second premolars, and 65% of maxillary second molars. Bilateral differences in the timing of permanent tooth eruption varied depending on type of tooth, and these differences tended to be smaller for teeth erupting at an earlier point in the order of eruption, and greater for those erupting at a later point. These results suggest that the failure of a contralateral tooth to erupt within 4 months of its counterpart might serve as a criterion in a diagnosis of suspected delayed eruption of a permanent tooth.