The Usefulness of Thin-section Iso-Voxel Diffusion Weighted Imaging for Stroke Subtype Classification: Case Series and Review.

BACKGROUND: Determining stroke subtypes on initial clinical evaluation is a prerequisite for the selection of appropriate initial treatment. Although diffusion-weighted imaging (DWI) is a powerful tool for detection of acute cerebral infarction, its diagnostic accuracy is not always sufficient particularly in the hyperacute phase. METHODS: Patients admitted within 2 weeks from the symptom onset with the diagnosis of acute ischemic strokes were analyzed with thin-section iso-voxel DWI, namely 3-dimension DWI (3D-DWI), to obtain axial, coronal, and sagittal sections in order to elucidate stroke characteristics. In this case series, we introduce the effectiveness of 3D-DWI. RESULTS: 3D-DWI uncovered stroke subtypes and distribution more precisely compared with conventional DWI. While previous studies indicated the utility of thin section DWI in detecting infratentrial infarctions, 3D-DWI is beneficial for the detection of not only infratentrial but also supratentorial lesions. Furthermore, since both 3D-DWI and magnetic resonance angiography (MRA) are multiplanar reconstruction images, the fusion image of 3D-DWI with MRA is available, enabling cross-reference of spatial cerebrovascular configuration and ischemic lesions. CONCLUSIONS: 3D-DWI is applicable to standard 1.5 T MRI by slight modification of data acquisition protocols, and becomes a key modality to solve the diagnostic puzzle of acute ischemic strokes.

FGF Suppresses Poldip2 Expression in Osteoblasts.

Osteoporosis is one of the most prevalent ageing-associated diseases that are soaring in the modern world. Although various aspects of the disease have been investigated to understand the bases of osteoporosis, the pathophysiological mechanisms underlying bone loss is still incompletely understood. Poldip2 is a molecule that has been shown to be involved in cell migration of vascular cells and angiogenesis. However, expression of Poldip2 and its regulation in bone cells were not known. Therefore, we examined the Poldip2 mRNA expression and the effects of bone regulators on the Poldip2 expression in osteoblasts. We found that Poldip2 mRNA is expressed in osteoblastic MC3T3-E1 cells. As FGF controls osteoblasts and angiogenesis, FGF regulation was investigated in these cells. FGF suppressed the expression of Poldip2 in MC3T3-E1 cells in a time dependent manner. Protein synthesis inhibitor but not transcription inhibitor reduced the FGF effects on Poldip2 gene expression in MC3T3-E1 cells. As for bone-related hormones, dexamethasone was found to enhance the expression of Poldip2 in osteoblastic MC3T3-E1 cells whereas FGF still suppressed such dexamethasone effects. With respect to function, knockdown of Poldip2 by siRNA suppressed the migration of MC3T3-E1 cells. Poldip2 was also expressed in the primary cultures of osteoblast-enriched cells and FGF also suppressed its expression. Finally, Poldip2 was expressed in femoral bone in vivo and its levels were increased in aged mice compared to young adult mice. These data indicate that Poldip2 is expressed in osteoblastic cells and is one of the targets of FGF. J. Cell. Biochem. 118: 1670-1677, 2017. © 2016 Wiley Periodicals, Inc.

Characterizing Genetic Transitions of Copy Number Alterations and Allelic Imbalances in Oral Tongue Carcinoma Metastasis.

Primary tumor (PT) heterogeneity can significantly affect the genetic profile of clones at metastatic sites. To understand the mechanisms underlying metastasis, we compared the genetic profile of paired PT and metastatic lymph node (MLN) samples obtained from patients with oral tongue squamous cell carcinoma (OTSCC). Large-scale genetic profiling was performed on paired PT-MLN samples obtained from 10 OTSCC patients using high-density single-nucleotide polymorphism microarrays. We compared the genetic profile of PT and MLN OTSCC samples to identify common and specific copy number alterations and copy-neutral loss-of-heterozygosity (CN-LOH). Unsupervised hierarchical clustering analysis indicated that 8 of the 10 PT-MLN sample pairs formed clusters, indicating that the primary and metastatic tumors were composed of predominantly genetically similar tumor cells. In 6 of the 10 pairs, 8q11.21, 8q12.2-3, and 8q21.3 gains, and 22q11.23 loss were detected in both the PT and MLN. In addition, 16p11.2 CN-LOH was identified in 9 of the 10 pairs. Conversely, 20q11.2 gain was only observed in the MLNs of 5 of the 10 sample pairs, indicating that genes in this chromosomal region may play a significant role in OTSCC lymph node metastasis. To confirm this, we investigated the expression of two candidate 20q11.2 genes in a separate patient cohort. The expression of one of these genes, E2F1, was significantly increased during the process of metastasis. This study indicates that additional genetic changes, such as 20q11.2 gain, which encodes the E2F1 gene, can be acquired through clonal evolution, and may be required for the metastatic process. © 2016 Wiley Periodicals, Inc.

Beta Adrenergic Receptor Stimulation Suppresses Cell Migration in Association with Cell Cycle Transition in Osteoblasts-Live Imaging Analyses Based on FUCCI System.

Osteoporosis affects over 20 million patients in the United States. Among those, disuse osteoporosis is serious as it is induced by bed-ridden conditions in patients suffering from aging-associated diseases including cardiovascular, neurological, and malignant neoplastic diseases. Although the phenomenon that loss of mechanical stress such as bed-ridden condition reduces bone mass is clear, molecular bases for the disuse osteoporosis are still incompletely understood. In disuse osteoporosis model, bone loss is interfered by inhibitors of sympathetic tone and adrenergic receptors that suppress bone formation. However, how beta adrenergic stimulation affects osteoblastic migration and associated proliferation is not known. Here we introduced a live imaging system, fluorescent ubiquitination-based cell cycle indicator (FUCCI), in osteoblast biology and examined isoproterenol regulation of cell cycle transition and cell migration in osteoblasts. Isoproterenol treatment suppresses the levels of first entry peak of quiescent osteoblastic cells into cell cycle phase by shifting from G1 /G0 to S/G2 /M and also suppresses the levels of second major peak population that enters into S/G2 /M. The isoproterenol regulation of osteoblastic cell cycle transition is associated with isoproterenol suppression on the velocity of migration. This isoproterenol regulation of migration velocity is cell cycle phase specific as it suppresses migration velocity of osteoblasts in G1 phase but not in G1 /S nor in G2 /M phase. Finally, these observations on isoproterenol regulation of osteoblastic migration and cell cycle transition are opposite to the PTH actions in osteoblasts. In summary, we discovered that sympathetic tone regulates osteoblastic migration in association with cell cycle transition by using FUCCI system.

Does Intraoral Miniplate Fixation Have Good Postoperative Stability After Sagittal Splitting Ramus Osteotomy? Comparison With Intraoral Bicortical Screw Fixation.

PURPOSE: Bicortical screw fixation systems and miniplate with monocortical screw fixation systems have been reported mainly in bilateral sagittal split ramus osteotomy (BSSO). This study compared postoperative stability between these 2 fixation systems by an intraoral approach. MATERIALS AND METHODS: This was a retrospective cohort study. The study sample was composed of patients treated by BSSO at the authors' institute from January 2006 through December 2012. All cases had facial symmetry and were performed by setback surgery. The predictor variable was treatment group (intraoral screw fixation [SG] vs intraoral miniplate fixation [MG]), and the primary outcome variable was stability defined as the change in the position of point B. Other outcome variables were stability defined as the change in the position of the menton, blood loss, incidence of postoperative temporomandibular joint disorder, and nerve injury. Descriptive and bivariate statistics were computed and the P value was set at .05. RESULTS: Seventy-five patients (35 men and 40 women; mean age, 25.8 yr) were divided into 2 groups (39 SG cases and 36 MG cases). Postoperative changes at point B and the menton in the 2 fixation groups were not statistically different. Lingual nerve injury occurred only in SG cases. Moreover, total blood loss was greater in SG cases. CONCLUSION: An intraoral miniplate with monocortical screw fixation system is recommended over intraoral bicortical screw fixation for bone segments in setback BSSO in patients without facial asymmetry.

Property of Human Bone Marrow Stromal Cells Derived From Bone Fragments Removed in Sagittal Split Ramus Osteotomy.

Bone tissue engineering is in the process of making the shift from bench to bed. Organ as a cell source is important for tissue engineering. The appropriate cells should be harvested without invasiveness and ethical problems. The authors focused on mandibular cortex bone fragments removed in sagittal split ramus osteotomy as a cell source for bone tissue engineering. These bone fragments were discarded after surgery until now. Bone marrow stromal cells (BMSCs) were harvested from inside of bone fragments, which is an endosteal region. Endosteal region is known to be a hematopoietic stem cell niche and harbors osteoblasts, preosteoblasts, and mesenchymal stem cells (MSCs). Bone marrow stromal cells could be cultured easily, and grew rapidly in vitro under ordinary serum-supplemented culture condition. The expression pattern of surface markers of BMSCs was the same as that of MSCs. Bone marrow stromal cells could differentiated into multiple mesenchymal lineages (osteoblasts, adipocytes, chondrocytes, and smooth muscle cells). These results indicated the existence of MSCs in BMSCs. The osteoblastic characters of BMSCs were examined more closely. Bone marrow stromal cells showed a high alkaline phosphatase activity, and expressed osteoblastic markers (PTHr, bone sialoprotein, Type I collagen, Rnut-related transcription factor 2, and osteocalcin). In transplantation experiments, BMSCs generated ectopic bone tissues on the border of hydroxyapatite scaffold without osteogenic differentiation-inducing agents such as dexamethasone (Dex) or bone morphogenetic protein. The results of this study suggest that mandibular cortex bone fragments removed in sagittal split ramus osteotomy are a good cell source for bone tissue engineering.

ß2 adrenergic receptor activation suppresses bone morphogenetic protein (BMP)-induced alkaline phosphatase expression in osteoblast-like MC3T3E1 cells.

ß adrenergic stimulation suppresses bone formation in vivo while its actions in osteoblastic differentiation are still incompletely understood. We therefore examined the effects of ß2 adrenergic stimulation on osteoblast-like MC3T3-E1 cells focusing on BMP-induced alkaline phosphatase expression. Morphologically, isoproterenol treatment suppresses BMP-induced increase in the numbers of alkaline phosphatase-positive small foci in the cultures of MC3T3-E1 cells. Biochemically, isoproterenol treatment suppresses BMP-induced enzymatic activity of alkaline phosphatase in a dose-dependent manner. Isoproterenol suppression of alkaline phosphatase activity is observed even when the cells are treated with high concentrations of BMP. With respect to cell density, isoproterenol treatment tends to suppress BMP-induced increase in alkaline phosphatase expression more in osteoblasts cultured at higher cell density. In terms of treatment protocol, continuous isoproterenol treatment is compared to cyclic treatment. Continuous isoproterenol treatment is more suppressive against BMP-induced increase in alkaline phosphatase expression than cyclic regimen. At molecular level, isoproterenol treatment suppresses BMP-induced enhancement of alkaline phosphatase mRNA expression. Regarding the mode of isoproterenol action, isoproterenol suppresses BMP-induced BRE-luciferase activity. These data indicate that isoproterenol regulates BMP-induced alkaline phosphatase expression in osteoblast-like MC3T3E1 cells.

Transforming growth factor-ß synthesized by stromal cells and cancer cells participates in bone resorption induced by oral squamous cell carcinoma.

Transforming growth factor beta (TGF-ß) plays a significant role in the regulation of the tumor microenvironment. To explore the role of TGF-ß in oral cancer-induced bone destruction, we investigated the immunohistochemical localization of TGF-ß and phosphorylated Smad2 (p-Smad2) in 12 surgical specimens of oral squamous cell carcinoma (OSCC). These studies revealed TGF-ß and p-Smad2 expression in cancer cells in all tested cases. Several fibroblasts located between cancer nests and resorbing bone expressed TGF-ß in 10 out of 12 cases and p-Smad2 in 11 out of 12 cases. Some osteoclasts also exhibited p âˆ¼ Smad2 expression. The OSCC cell line, HSC3, and the bone marrow-derived fibroblastic cell line, ST2, synthesized substantial levels of TGF-ß. Culture media derived from HSC3 cells could stimulate Tgf-ß1 mRNA expression in ST2 cells. Recombinant TGF-ß1 could stimulate osteoclast formation induced by receptor activator of nuclear factor kappa-B ligand (RANKL) in RAW264 cells. TGF-ß1 could upregulate the expression of p-Smad2 in RAW264 cells, and this action was suppressed by the addition of a neutralizing antibody against TGF-ß or by SB431542. Transplantation of HSC3 cells onto the calvarial region of athymic mice caused bone destruction, associated with the expression of TGF-ß and p-Smad2 in both cancer cells and stromal cells. The bone destruction was substantially inhibited by the administration of SB431542. The present study demonstrated that TGF-ß synthesized by both cancer cells and stromal cells participates in the OSCC-induced bone destruction.

Comprehensive treatment approach for bilateral cleft lip and palate in an adult with premaxillary osteotomy, tooth autotransplantation, and 2-jaw surgery.

We report the successful treatment of a woman aged 25 years 3 months with bilateral cleft lip and palate. She had a protruded premaxilla, collapsed posterior segments, wide alveolar defects with oronasal fistulae, a congenital missing tooth, and severe facial asymmetry with a transverse occlusal cant. The comprehensive treatment approach included (1) premaxillary osteotomy combined with alveolar bone grafting to reposition the premaxilla and minimize the wide alveolar defects, (2) autotransplantation of a tooth with complete root formation to the grafted bone region to restore the missing tooth without a prosthesis such as a dental implant or bridge, and (3) 2-jaw surgery to improve facial asymmetry. The premaxillary osteotomy was managed orthodontically, in combination with bone grafting. The results suggest that surgical orthodontic treatment with tooth autotransplantation might be useful to improve the occlusion and facial esthetics without prosthetics.

CCN3 protein participates in bone regeneration as an inhibitory factor.

CCN3, a member of the CCN protein family, inhibits osteoblast differentiation in vitro. However, the role of CCN3 in bone regeneration has not been well elucidated. In this study, we investigated the role of CCN3 in bone regeneration. We identified the Ccn3 gene by microarray analysis as a highly expressed gene at the early phase of bone regeneration in a mouse bone regeneration model. We confirmed the up-regulation of Ccn3 at the early phase of bone regeneration by RT-PCR, Western blot, and immunofluorescence analyses. Ccn3 transgenic mice, in which Ccn3 expression was driven by 2.3-kb Col1a1 promoter, showed osteopenia compared with wild-type mice, but Ccn3 knock-out mice showed no skeletal changes compared with wild-type mice. We analyzed the bone regeneration process in Ccn3 transgenic mice and Ccn3 knock-out mice by microcomputed tomography and histological analyses. Bone regeneration in Ccn3 knock-out mice was accelerated compared with that in wild-type mice. The mRNA expression levels of osteoblast-related genes (Runx2, Sp7, Col1a1, Alpl, and Bglap) in Ccn3 knock-out mice were up-regulated earlier than those in wild-type mice, as demonstrated by RT-PCR. Bone regeneration in Ccn3 transgenic mice showed no significant changes compared with that in wild-type mice. Phosphorylation of Smad1/5 was highly up-regulated at bone regeneration sites in Ccn3 KO mice compared with wild-type mice. These results indicate that CCN3 is up-regulated in the early phase of bone regeneration and acts as a negative regulator for bone regeneration. This study may contribute to the development of new strategies for bone regeneration therapy.

Maxillary distraction osteogenesis for treatment of cleft lip and palate in a patient with X-linked agammaglobulinemia.

X-linked agammaglobulinemia (XLA) is a congenital immune deficiency disorder caused by abnormal antibody production. It is a rare disease with an estimated frequency of 1 in 379,000 that has X-linked recessive heredity and develops only in males. The clinical problems include bacterial infection such as otitis media, sinusitis, and bronchitis. In recent years it has become possible to diagnose XLA in the early stage and intravenous immunoglobulin replacement therapy has permitted survival to adulthood. However, there have been no reports of oral surgery in patients with XLA. Here, we describe a case in which immunoglobulin replacement therapy given pre- and postoperatively was used to control infection in oral surgery and maxillary distraction osteogenesis performed for improving occlusion and appearance of a cleft lip and palate in a patient with XLA.

Soft tissue cephalometric norms for orthognathic and cosmetic surgery.

PURPOSE: Proportionality of the lower and middle thirds of the face is a key determinant of successful orthognathic treatment. A flatter profile and marked variance of the soft tissue envelope in the Japanese population complicates the accurate assessment of these proportions. This study aimed to identify gender differences and establish norms for Japanese young adults using the method of soft tissue cephalometric analysis (STCA) by Arnett et al (Am J Orthod Dentofacial Orthop 116:239, 1999). MATERIALS AND METHODS: Lateral cephalograms of 49 young normal Japanese subjects (19 men, 30 women) were selected from the archival records and analyzed with STCA. The Student t test was used to compare mean values of the male and female groups. RESULTS: Significant differences were found between women and men. Men had a flatter occlusal plane and a more acute nasolabial angle than women. Men showed larger values for upper and lower lip thickness, menton soft tissue thickness, and vertical face length, especially in the lower third of the face. Women had a more projected midface than men. Compared with established STCA norms, the Japanese have more midfacial projection. CONCLUSIONS: Significant gender differences were found in the thickness, lower third length, and midface projection in Japanese young adults, which should be taken into account when interpreting measurements for orthognathic surgical planning. These differences can serve as norms for STCA in young Japanese adults. Differences were noted between the reference values of Arnett et al and Japanese subjects.

Unusual expression of red fluorescence at M phase induced by anti-microtubule agents in HeLa cells expressing the fluorescent ubiquitination-based cell cycle indicator (Fucci).

Plinabulin (NPI-2358) is a novel microtubule-depolymerizing agent. In HeLa cells, plinabulin arrests the cell-cycle at M phase and subsequently induces mitotic catastrophe. To better understand the effects on this compound on the cell-cycle, we used the fluorescent ubiquitination-based cell cycle indicator (Fucci), which normally enables G1 and S/G2/M cells to emit red and green fluorescence, respectively. When HeLa-Fucci cells were treated with 50 nM plinabulin, cells began to fluoresce both green and red in an unusual pattern; most cells exhibited the new pattern after 24 h of treatment. X-irradiation efficiently induced G2 arrest in plinabulin-treated cells and significantly retarded the emergence of the unusual pattern, suggesting that entering M phase is essential for induction of the pattern. By simultaneously visualizing chromosomes with GFP-histone H2B, we established that the pattern emerges after nuclear envelope breakdown but before metaphase. Pedigree assay revealed a significant relationship between the unusual expression and mitotic catastrophe. Nocodazole, KPU-133 (a more potent derivative of plinabulin), and paclitaxel also exerted similar effects. From these data, we conclude that the unusual pattern may be associated with dysregulation of late M phase-specific E3 ligase activity and mitotic catastrophe following treatment with anti-microtubule agents.

CXCL2 synthesized by oral squamous cell carcinoma is involved in cancer-associated bone destruction.

To explore the mechanism of bone destruction associated with oral cancer, we identified factors that stimulate osteoclastic bone resorption in oral squamous cell carcinoma. Two clonal cell lines, HSC3-C13 and HSC3-C17, were isolated from the maternal oral cancer cell line, HSC3. The conditioned medium from HSC3-C13 cells showed the highest induction of Rankl expression in the mouse stromal cell lines ST2 and UAMS-32 as compared to that in maternal HSC3 cells and HSC3-C17 cells, which showed similar activity. The conditioned medium from HSC3-C13 cells significantly increased the number of osteoclasts in a co-culture with mouse bone marrow cells and UAMS-32 cells. Xenograft tumors generated from these clonal cell lines into the periosteal region of the parietal bone in athymic mice showed that HSC3-C13 cells caused extensive bone destruction and a significant increase in osteoclast numbers as compared to HSC3-C17 cells. Gene expression was compared between HSC3-C13 and HSC3-C17 cells by using microarray analysis, which showed that CXCL2 gene was highly expressed in HSC3-C13 cells as compared to HSC3-C17 cells. Immunohistochemical staining revealed the localization of CXCL2 in human oral squamous cell carcinomas. The increase in osteoclast numbers induced by the HSC3-C13-conditioned medium was dose-dependently inhibited by addition of anti-human CXCL2-neutralizing antibody in a co-culture system. Recombinant CXCL2 increased the expression of Rankl in UAMS-32 cells. These results indicate that CXCL2 is involved in bone destruction induced by oral cancer. This is the first report showing the role of CXCL2 in cancer-associated bone destruction.

Mandibular distraction osteogenesis in a skeletal Class II patient with obstructive sleep apnea.

We report the orthodontic treatment of a 31-year-old man with severe skeletal Class II malocclusion and documented obstructive sleep apnea (OSA). He had a retrognathic profile with an overbite of 4 mm and an overjet of 14 mm. Mandibular distraction osteogenesis was performed to lengthen the small, retruded mandible by 18 mm and improve the symptoms of OSA. Orthodontic treatment after the mandibular distraction osteogenesis procedure lasted 3 years 1 month. An acceptable occlusion was obtained, and the patient's OSA was significantly alleviated. Although the patient was satisfied with the treatment, condylar resorption was observed. The relevance of condylar resorption with reference to a comprehensive evaluation of the treatment outcome is discussed.

A case of amyloid myopathy diagnosed during the treatment of myopathy associated with anti-signal recognition particle antibodies.

A 78-year-old man presented with subacute progressive proximal weakness and dysphagia. A biopsy specimen from the left biceps femoris revealed evidence of necrotic and regenerating muscle fibers, but lymphocyte infiltration was not noted. The patient was diagnosed with necrotizing myopathy with anti-signal recognition particle (SRP) antibodies. Concomitant therapy with prednisolone and azathioprine caused the serum CK level to return to normal and it caused clinical manifestations to abate. One year later, however, muscle weakness worsened. Immunoelectrophoresis of serum revealed IgG M protein, and muscle pathology revealed amyloid deposits in numerous blood vessels and at the periphery of a few muscle fibers, and deposits stained positive for anti-λ light chain antibody. The patient was diagnosed with amyloid myopathy, and therapy for systemic amyloid light chain amyloidosis caused muscle weakness to diminish. Amyloidosis is believed to be the primary pathology in this case based on the patient's response to treatment reaction, but the significance of a case involving both amyloid myopathy and necrotizing myopathy warranted examination.

Effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) on bone consolidation on distraction osteogenesis: a preliminary study in rabbit mandibles.

AIM: To examine the effectiveness of administering recombinant human bone morphogenetic protein-2 (rhBMP-2) in distraction osteogenesis. MATERIAL AND METHODS: Twenty-one mature male Japanese white rabbits underwent unilateral mandibular osteotomy. After 5 days, the osteotomized mandibles were distracted 1mm/day for 10 days. On the first day (groups A-1 [n=4] and A-2 [n=4]) or on the last day (group B [n=5]) of distraction, rhBMP-2 mixed with collagen gel was injected into the distraction zone. In control groups (groups C-1 [n=4] and C-2 [n=4]), the mandibles were distracted without rhBMP-2 injection. At the end of the distraction period (groups A-1 and C-1) and after 2 weeks of consolidation (groups A-2, B, and C-2), the distracted mandibles were harvested and examined with soft radiographs, peripheral quantitative computed tomography (pQCT), and microscopy. RESULTS: Radioopacity was more marked in the distraction zone of the groups with rhBMP-2 than in control groups. The mineral density of the cortical bone (BMD) was higher in groups B and A-2 than in group C-2. Histologically, bone formation was more advanced in groups A-2 and B than in group C-2. The cortical BMD was higher in group A-1 than in group C-1. Histologically, bone formation was more advanced in groups A-2 and B than in group C-2. CONCLUSION: These results suggest that rhBMP-2 promotes bone formation in distraction osteogenesis.