Taku Kojima

A histological assessment on the distribution of the osteocytic lacunar canalicular system using silver staining.

Giving the complexity that characterizes the mechanisms of bone remodeling and the number of events that have to be in absolute harmony for it to occur flawlessly, the postulation that temporospatial distribution of osteocytes and their lacunar canalicular system might influence and be influenced by bone remodeling can be regarded, at least, as feasible. In this study, using Schoens silver staining, we have examined the distribution of the osteocytic lacunar canalicular system (OLCS) in bones of developing mice. Trabecular bones of 3-day-old, 2-week-old, and 3-week-old mice displayed osteocytic cytoplasmic processes without any perceptible alignment. Also, many plump osteocytes were embedded in the mineralized bone matrix in a disorderly manner. At 4 weeks of age, however, mice bones showed some osteocytic processes that reached the bone surface on a right angle, while other osteocytes displayed the same features seen on 3-week specimens. Samples at 8 weeks of age featured osteocytes with their usual spindle shape, organized so as to parallel the longitudinal axis of trabecular bone. They also extended their cytoplasmic processes perpendicularly to the bone surface. However, several osteocytes immersed in older bone, i.e., a residual mix of cartilage and bone matrices, still showed a random pattern of distribution of their cytoplasmic processes. Up to 12 weeks of age, the majority of the osteocytes became flattened and were shown to be aligned with their long axis paralleling the bone surface. This tendency for such a gradual arrangement was also observed in cortical bones. We have further demonstrated that 8-week-old osteoprotegerin-deficient mice, which demonstrated histological evidence of higher than average bone turnover, revealed a disorganized OLCS. Given the data gathered in this work, the OLCS appears to assume an organized, probably function-related spatial distribution as normal bone remodeling goes on.

Effects of orthognathic surgery on psychological status of patients with jaw deformities

The purpose of this study was to determine the effect of orthognathic surgery on psychological status. The subjects were 119 patients (38 males and 81 females, mean age 25.5±9.4 years) who underwent orthognathic surgery. They were divided into class III (84 patients), class II (20 patients), and class I (15 patients) groups according to the anteroposterior skeletal pattern, and they were also divided into an asymmetry group (51 patients) and a symmetry group (68 patients). We assessed psychological status using the Minnesota Multiphasic Personality Inventory (MMPI) before surgery and at more than 6 months after surgery. The MMPI scores for the depression, hysteria, psychasthenia, and social introversion scales were significantly higher than standard values before surgery, and the hypomania scale significantly lower. The cannot say scale, depression scale, and hysteria scale decreased significantly after surgery. A comparison of MMPI scores among the groups showed the depression scale in the class III group to be higher than those in the class I and II groups; there was no significant difference between the asymmetry and symmetry groups. In conclusion, orthognathic surgery has a positive influence on the psychological status of patients with jaw deformities, especially patients with skeletal class III malocclusion.

Keratinocytes of Tissue-Engineered Human Oral Mucosa Promote Re-Epithelialization After Intraoral Grafting in Athymic Mice

PURPOSE The objective of this study was to investigate the role of grafted oral keratinocytes in a transplanted ex vivo-produced oral mucosa equivalent (EVPOME) in the regeneration and/or healing process of the oral mucosa at the recipient site. MATERIALS AND METHODS The EVPOME was developed in a serum-free defined culture system without a feeder layer. EVPOME is composed of a stratified layer of human oral keratinocytes that are seeded onto a human cadaveric dermis, AlloDerm (LifeCell, Branchburg, NJ). Intraorally grafted EVPOMEs in athymic mice (BALB/c) were excised, contiguous with the surrounding oral mucosa, on days 5, 7, 14, and 21 after grafting. Serial sections were stained with hematoxylin-eosin and immunohistochemically analyzed for cytokeratin 17 (CK17) expression to distinguish the human-cultured EVPOME epithelial keratinocytes from murine oral keratinocytes. RESULTS All EVPOME epithelial cells showed intense immunoreactivity for CK17, whereas mouse buccal mucosal epithelial cells did not show CK17 immunoreactivity. The grafted EVPOME maintained a stratified epithelial layer for up to 5 days after grafting. By day 7 after grafting, a portion of the EVPOME epithelial layer peeled away from the AlloDerm, and a thin, CK17-immunonegative epithelial layer extended from the adjacent thick epithelial layer of the mouse and contacted the CK17-immunopositive EVPOME epithelium. From days 14 to 21 after grafting, the stratification of the CK17-immunonegative continuous mouse epithelium increased compared with earlier time points and showed a similar appearance to the epithelium of the adjacent mouse mucosa. In contrast, no epithelial coverage of the AlloDerm that was grafted without keratinocytes was observed for up to 21 days after grafting. The grafted AlloDerm without cells resulted in tissue necrosis that was accompanied by a dramatic infiltration of inflammatory cells by day 14. CONCLUSIONS These findings suggest that grafting of EVPOME with viable oral keratinocytes onto an intraoral mucosal wound plays an active role in promotion of re-epithelialization of the oral wound during the subsequent healing process.

FGFR3 down-regulates PTH/PTHrP receptor gene expression by mediating JAK/STAT signaling in chondrocytic cell line

The signaling axis comprising the parathyroid hormone (PTH)-related peptide (PTHrP), the PTH/PTHrP receptor and the fibroblast growth factor receptor 3 (FGFR3) plays a central role in chondrocyte proliferation. The Indian hedgehog (IHH) gene is normally expressed in early hypertrophic chondrocytes, and its negative feedback loop was shown to regulate PTH/PTHrP receptor signaling. In this study, we examined the regulation of PTH/PTHrP receptor gene expression in a FGFR3-transfected chondrocytic cell line, CFK2. Expression of IHH could not be verified on these cells, with consequent absence of hypertrophic differentiation. Also, expression of the PTH/PTHrP receptor (75% reduction of total mRNA) and the PTHrP (50% reduction) genes was reduced in CFK2 cells transfected with FGFR3 cDNA. Interestingly, we verified significant reduction in cell growth and increased apoptosis in the transfected cells. STAT1 was detected in the nuclei of the CFK2 cells transfected with FGFR3 cDNA, indicating predominance of the JAK/STAT signaling pathway. The reduction in PTH/PTHrP receptor gene in CFK2 cells overexpressing FGFR3 was partially blocked by treatment with an inhibitor of JAK3 (WHI-P131), but not with an inhibitor of MAPK (SB203580) or JAK2 (AG490). Altogether, these findings suggest that FGFR3 down-regulates PTH/PTHrP receptor gene expression via the JAK/STAT signaling in chondrocytic cells.

Histological examination of bone regeneration achieved by combining grafting with hydroxyapatite and thermoplastic bioresorbable plates

In this study, we present a novel guided bone regeneration (GBR) concept that consists of combining Boneject, a bone substitute containing atelocollagen and bovine hydroxyapatite particles, with thermoplastic, bioresorbable plates (DeltaSystem) known to resist mechanical loading. In rat calvariae, standardized bone defects were filled with Boneject and covered with a convex DeltaSystem plate. Tissue from rats at 1, 2, 4, 8, and 12 weeks postoperation were fixed with an aldehyde solution, and the new bone formed at the defects was histologically assessed. At 1 week, alkaline phosphatase (ALP)-negative cells deriving from the bottom region of the defect could be found up to half the height of the cavity. Boneject particle surfaces in the bottom region revealed an intense osteopontin immunopositivity whereas those in the upper region did not. Tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts accumulated on the surfaces of osteopontin-coated particles. A newly formed, woven-like bone featuring ALP-positive osteoblasts extended from the native bone. At the second week, the newly formed woven bone had surrounded the Boneject particles. Cement lines, which indicate active bone remodeling, could be observed in the new bone despite its immaturity. Four weeks after surgery, the new bone had reached the height of the DeltaSystem plate, and just beneath it a periostin-positive fibrous layer covered the mix of new bone and Boneject particles. By then, despite having acceptable histological features, electron probe microanalyzer (EPMA) and transmission electron microscope (TEM) analyses revealed that the new bone could not be regarded as compact bone. At 8 and 12 weeks, the new bone showed compact bone-like features according to TEM and EPMA assessments. Summarizing, the combination of a bone substitute such as Boneject and a rigid, bioresorbable plate appears to be osteoconductive and to promote bone remodeling, leading to the genesis of a tissue similar to the one that is regarded as the “gold standard” for bone regeneration: the compact bone.

A histologic study of deformation of the mandibular condyle caused by distraction in a rat model

OBJECTIVE This study investigated the bone resorption process of the rat mandibular condyle after mandibular distraction. STUDY DESIGN Male Wistar rats at 10 weeks of age underwent unilateral mandibular distraction at 0.175 mm per 12 hours for 10 days. Histologic and histochemical analyses were performed at postoperative day 1 and weeks 1 and 3. RESULTS High-resolution computed tomography (micro-CT) observations showed that deformation of the condyle occurred in the anterior region, where a discontinuity of the condylar cartilage layer was found in histologic sections. This destroyed area gathered many osteoclasts. In the central region, disorganization with a thin hypertrophic cell layer was recognizable by day 1 but later thickened. Morphologic recovery of the mandibular condyle could be attained by week 3 in this animal model. CONCLUSIONS These morphologic findings indicate that rapid deformation of the condyle, with destruction of the cartilage layer and bone resorption, was caused by artificial distraction.

Ultrastructural observation on cells meeting the histological criteria for preosteoblasts--a study in the mouse tibial metaphysis.

Preosteoblasts are currently defined as the precursors of mature osteoblasts. These cells are morphologically diverse and may represent a continuum during osteoblast differentiation. We have attempted to categorize the different preosteoblastic phenotypes in vivo by examining bone cells expressing the runt-related transcription factor 2, alkaline phosphatase and BrdU incorporation - histological traits of a preosteoblast - under transmission electron microscopy (TEM). TEM observations demonstrated, at least, in part two preosteoblastic subtypes: (i) a cell rich in cisterns of rough endoplasmic reticulum (rER) with vesicles and vacuoles and (ii) a subtype featuring extended cytoplasmic processes that connect with distant cells, with a small amount of scattered cisterns of rER and with many vesicles and vacuoles. ER-rich cells, whose cellular machinery is similar to that of an osteoblast, were often seen adjacent to mature osteoblasts, and therefore, may be ready for terminal differentiation. In contrast, ER-poor and vesicle-rich cells extended their cytoplasmic processes to mature osteoblasts and, frequently, to bone-resorbing osteoclasts. The abundant vesicles and vacuoles identified in this cell type indicate that this cell is involved in vesicular transport rather than matrix synthesis activity. In summary, our study verified the morphological diversity and the ultrastructural properties of osteoblastic cells in vivo.

A simple technique for repositioning of the mandible by a surgical guide prepared using a three-dimensional model after segmental mandibulectomy

BackgroundMandibular reconstruction is performed after segmental mandibulectomy, and precise repositioning of the condylar head in the temporomandibular fossa is essential for maintaining preoperative occlusion.MethodsIn cases without involvement of soft tissue around the mandibular bone, the autopolymer resin in a soft state is pressed against the lower border of the mandible and buccal and lingual sides of the 3D model on the excised side. After hardening, it is shaved with a carbide bar to make the proximal and distal parts parallel to the resected surface in order to determine the direction of mandibular resection. On the other hand, in cases that require resection of soft tissue around the mandible such as cases of a malignant tumor, right and left mandibular rami of the 3D model are connected with the autopolymer resin to keep the preoperative position between proximal and distal segments before surgical simulation. The device is made to fit the lower border of the anterior mandible and the posterior border of the mandibular ramus. The device has a U-shaped handle so that adaptation of the device will not interfere with the soft tissue to be removed and has holes to be fixed on the mandible with screws.ResultsWe successfully performed the planned accurate segmental mandibulectomy and the precise repositioning of the condylar head by the device.ConclusionsThe present technique and device that we developed proved to be simple and useful for restoring the preoperative condylar head positioning in the temporomandibular fossa and the precise resection of the mandible.

III–1 Vertical Distraction Osteogenesis of a Reconstructed Mandible with a Free Non-Vascularized Iliac Bone Garft Combined with LIPUS Treatment: A Case Report

Introduction: We report a patient who underwent vertical distraction osteogenesis of a reconstructed mandible. Treatment involved a free non-vascularized iliac bone graft combined with LIPUS treatment, followed by dental implant treatment. Case: The patient was a 37-year-old female who underwent mandibular resection for treatment of a benign ameloblastoma. The postoperative course was uneventful, and dental implant treatment was planned in the reconstructed mandible. However, bone height in the reconstructed mandible was insufficient for ideal prosthetic rehabilitation. Therefore, we performed vertical distraction osteogenesis of the reconstructed mandible, resulting in a total distraction length of 14 mm. LIPUS was performed to accelerate bone healing in the distracted area for 3 months during the consolidation period. After that, there was a bone defect between the native bone and the mesial side of the transport segment. The patient underwent grafting of bone harvested from the distal ridge of the transport segment and the mandibular ramus, to obtain sufficient volume of the alveolar ridge. Bone was held in position with titanium mesh. CT images demonstrated that density of the iliac bone in the reconstructed mandible was sufficient for implant treatment. Eventually, the patient received dental implant prosthetic rehabilitation in the reconstructed mandible. Discussion and Conclusion: Bone density of an iliac bone graft is often less than that of native bone. In this case, we performed vertical distraction osteogenesis combined with LIPUS to generate sufficient bone height and density. The patient successfully received adequate esthetics and function of the implant-supported prosthesis.