Hiroaki Ichioka

Effects of mechanical stress on cytokine production in mandible-derived osteoblasts.

OBJECTIVE Mechanical stress is known to be an important factor in the regulation of bone remodeling, and mandibular bone is continuously exposed to mechanical stressors such as occlusal force. Therefore, in this study, we investigated the effects of mechanical stress approaching occlusal force, to which mandible-derived osteoblasts (MDOB) are exposed, on cytokine expression and production using an original hydrostatic pressure apparatus. MATERIALS AND METHODS The levels of cytokine in MDOB were examined by real-time RT-PCR, ELISA, and western blotting. In addition, mitogen-activated protein kinase inhibitor for ERK1/2, JNK, and p-38 pathways was used to identify the signal transduction pathway. RESULTS Hydrostatic pressure increased the expression of IL-6 and TNF-α mRNA in a magnitude- and time-dependent manner and also enhanced IL-6 and TNF-α protein production. Furthermore, hydrostatic pressure changed the RANKL/OPG ratio in favor of RANKL for both mRNA and protein levels. Specific inhibitor of p-38 pathway but not that of the ERK1/2 and JNK pathways suppressed the up-regulation of RANKL production induced by hydrostatic pressure loading. CONCLUSION These results suggest that MDOB play a role in cytokine production in response to mechanical stress and that occlusal force may support the maintenance of mandible bone homeostasis by activating bone remodeling through osteoclastogenesis.

β-cryptoxanthin regulates bone resorption related-cytokine production in human periodontal ligament cells

OBJECTIVE β-cryptoxanthin (β-cry) is a type of carotenoid found in certain fruits and vegetables. Although it has been shown that β-cry inhibits alveolar bone resorption, the molecular mechanisms for this have not yet been clarified. In the present study, we investigated the effects of β-cry on bone resorption related-cytokine production in human periodontal ligament (hPDL) cells. DESIGN hPDL cells were stimulated with β-cry (1×10(-7)mol/l), mechanical stress (1 or 6MPa), and P. gingivalis. The production of interleukin (IL)-1β, IL-6, IL-8, tumour necrosis factor (TNF)-α, osteoprotegerin (OPG), and receptor activator of nuclear factor kappa-B ligand (RANKL) were analyzed by RT-PCR and ELISA. RESULTS The production of IL-1β, IL-6, IL-8, and TNF-α was not induced in hPDL cells after stimulation with β-cry, although these cytokines were produced after stimulation with P. gingivalis. On the other hand, IL-6 and IL-8 were produced after exposure to 6MPa of mechanical stress. The production of IL-6 and IL-8 was significantly decreased by the addition of β-cry. Furthermore, β-cry up-regulated the production of OPG, but not RANKL. CONCLUSION β-cry inhibited the production of IL-6 and IL-8 induced by mechanical stress and periodontopathogenic bacteria in hPDL cells. Moreover, β-cry up-regulated OPG production. These results suggest that β-cry may prevent bone resorption in periodontitis.

Vibrational algorithms for quantitative crystallographic analyses of hydroxyapatite-based biomaterials: I, theoretical foundations

The Raman spectroscopic method has quantitatively been applied to the analysis of local crystallographic orientation in both single-crystal hydroxyapatite and human teeth. Raman selection rules for all the vibrational modes of the hexagonal structure were expanded into explicit functions of Euler angles in space and six Raman tensor elements (RTE). A theoretical treatment has also been put forward according to the orientation distribution function (ODF) formalism, which allows one to resolve the statistical orientation patterns of the nm-sized hydroxyapatite crystallite comprised in the Raman microprobe. Close-form solutions could be obtained for the Euler angles and their statistical distributions resolved with respect to the direction of the average texture axis. Polarized Raman spectra from single-crystalline hydroxyapatite and textured polycrystalline (teeth enamel) samples were compared, and a validation of the proposed Raman method could be obtained through confirming the agreement between RTE values obtained from different samples.

Generation of Directly Converted Human Osteoblasts That Are Free of Exogenous Gene and Xenogenic Protein

Generation of osteoblasts from human somatic cells may be applicable in an effective transplantation therapy against bone diseases. Recently we established a procedure to directly convert human fibroblasts into osteoblasts by transducing some transcription factor genes via retroviral vectors. However, retroviral vector‐mediated transduction may potentially cause tumor formation from the infected cells, thus a non‐viral gene transfection method may be more preferable for preparation of osteoblasts to be used for transplantation therapy. Here, we constructed a plasmid vector encoding Oct4, Osterix, and L‐Myc that were an appropriate combination of transcription factors for this purpose. Osteoblast‐like phenotypes including high alkaline phosphatase (ALP) activity, bone matrix production and osteoblast‐specific gene expression were induced in normal human fibroblasts that were transfected with the plasmid followed by culturing in osteogenic medium. The plasmid‐driven directly converted osteoblasts (p‐dOBs) were obtained even in the absence of a xenogenic protein. The plasmid vector sequence had fallen out of the p‐dOBs. The cells formed deposition of calcified bodies in situ after transplantation into mice. These results strongly suggest that p‐dOBs can be put into practical use for a novel cell‐based therapy against bone diseases. J. Cell. Biochem. 117: 2538–2545, 2016.

Vibrational algorithms for quantitative crystallographic analyses of hydroxyapatite-based biomaterials: II, application to decayed human teeth

A systematic investigation, based on highly spectrally resolved Raman spectroscopy, was undertaken to research the efficacy of vibrational assessments in locating chemical and crystallographic fingerprints for the characterization of dental caries and the early detection of non-cavitated carious lesions. Raman results published by other authors have indicated possible approaches for this method. However, they conspicuously lacked physical insight at the molecular scale and, thus, the rigor necessary to prove the efficacy of this spectroscopy method. After solving basic physical challenges in a companion paper, we apply them here in the form of newly developed Raman algorithms for practical dental research. Relevant differences in mineral crystallite (average) orientation and texture distribution were revealed for diseased enamel at different stages compared with healthy mineralized enamel. Clear spectroscopy features could be directly translated in terms of a rigorous and quantitative classification of crystallography and chemical characteristics of diseased enamel structures. The Raman procedure enabled us to trace back otherwise invisible characteristics in early caries, in the translucent zone (i.e., the advancing front of the disease) and in the body of lesion of cavitated caries.

Comparison of postmortem butane distribution between two fatal butane poisoning cases

Fatal accidents during butane abuse frequently occur in Japan and in many countries around the world. Although analytical data about butane concentration in postmortem samples is being accumulated, when using the data to determine the cause of death, careful interpretation is required because the gas is easily diffused. Two fatal butane poisoning cases were encountered, and butane quantification of autopsy samples obtained from left and right heart blood, femoral blood, kidney, liver, lung, brain and fatty tissues was performed. In both cases, butane concentration in the left heart blood was lower than in the right heart blood or the femoral blood, despite gas inhalation. These findings may indicate that the deceased individuals interrupted gas inhalation and inhaled room air immediately before their death, therefore ruling out asphyxia due to anoxia as the mechanism of death. Case 1, which was suspected to be a not acute death, showed a very high butane concentration ratio of fatty tissues to femoral blood of over 70. Case 2 was considered an acute death, and the butane concentration ratio of fatty tissues to femoral blood was 8.2. These results are consistent with previously reported findings showing that much higher ratios of fatty tissues to blood are compatible with long survival time. In conclusion, the comparison of butane concentration among different samples, including left heart blood versus right heart blood and fatty tissues versus blood, is useful when interpreting the result of postmortem butane analysis to examine the mechanism of death and survival time.

Infant fatality case with excessive chylous ascites

An 11-month-old boy with marked abdominal distension was found dead in the prone position at home. Since there were many bruises in the non-protruding regions of the head, face, and abdomen, a medicolegal autopsy was performed the following day. The boy was smaller than average (height: 68.5 cm; weight: 7.8 kg); his extremities were thin; and his abdomen was remarkably bulging. Chylous ascites (1600 mL) was observed in the peritoneal cavity and chylous pleural effusion (left: 5 mL; right: 10 mL) in the thoracic cavity. A fibrous induration, approximately 2.0 × 1.5 cm in size, was observed in the root of the small bowel mesentery. Congenital chylothorax and chylous ascites were suspected. However, the remarkably withered thymus and an old injury in the superior labial frenulum suggested that the chylous ascites may have been further deteriorated by injuries sustained during physical abuse. Examination suggested that the death was sudden. Thus, we inferred that the cause of death was circulatory and respiratory failure due to excessive chylous ascites. Among the reported cases of chylous ascites in pediatric patients, some patients experiencing abuse were identified on the basis of their chief complaints of vomiting or abdominal distension. Medical and child welfare staff should be made aware of this information.

Comparison between Temperature Gradient Gel Electrophoresis of Bacterial 16S rDNA and Diatom Test for Diagnosis of Drowning

When a body is discovered in water, it is difficult to conclude whether the cause of death was drowning, even today. Although diatom testing by the digestive method is classical, we hypothesized that aquatic bacteria, as well as diatoms, might be detected in drowned bodies, and conducted temperature gradient gel electrophoresis (TGGE)‐targeting 16S rDNA. DNA was extracted from the site water, and from heart blood and liver samples from 27 bodies concluded as drowning deaths by autopsy and subjected to TGGE after amplification of 16S rDNA by polymerase chain reaction. We observed whether the feature point of each 16S rDNA from the site water and blood or liver samples matched. Considerably higher correspondence was observed in drowned bodies, and the rate was higher than that achieved with the digestive method. Moreover, TGGE is safer than the digestive method. Our study suggests that this method can aid diagnosis of drowning.

Vibrational monitor of early demineralization in tooth enamel after in vitro exposure to phosphoridic liquid.

The Raman spectroscopic method has been applied to quantitatively assess the in vitro degree of demineralization in healthy human teeth. Based on previous evaluations of Raman selection rules (empowered by an orientation distribution function (ODF) statistical algorithm) and on a newly proposed analysis of phonon density of states (PDOS) for selected vibrational modes of the hexagonal structure of hydroxyapatite, a molecular-scale evaluation of the demineralization process upon in vitro exposure to a highly acidic beverage (i.e., CocaCola™ Classic, pH=2.5) could be obtained. The Raman method proved quite sensitive and spectroscopic features could be directly related to an increase in off-stoichiometry of the enamel surface structure since the very early stage of the demineralization process (i.e., when yet invisible to other conventional analytical techniques). The proposed Raman spectroscopic algorithm might possess some generality for caries risk assessment, allowing a prompt non-contact diagnostic practice in dentistry.

Electrical stimulation with periodic alternating intervals stimulates neuronal cells to produce neurotrophins and cytokines through activation of mitogen-activated protein kinase pathways.

Peripheral neuropathy is a representative complication of dental surgery. Electrical therapy, based on electrical stimulation with periodic alternating intervals (ES-PAI), may promote nerve regeneration after peripheral nerve injury in a non-invasive manner, potentially providing an effective therapy for neuropathy. This study aimed to analyze the molecular mechanisms underlying the nerve recovery stimulated by ES-PAI. In brief, ES-PAI was applied to a neuronal cell line, Neuro2A, at various intensities using the pulse generator apparatus, FREUDE. Cell viability, neurotrophin mRNA expression, and cytokine production were examined using a tetrazolium-based assay, real-time RT-PCR, and ELISA, respectively. Mitogen-activated protein kinase (MAPK) signaling was assessed using flow cytometry. It was found that ES-PAI increased the viability of cells and elevated expression of nerve growth factor (NGF) and neurotrophin-3 (NT-3); ESPAI also augmented vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) expression, which was restored by addition of p38 inhibitors. Phosphorylation of p38 and extracellular signal-regulated kinase 1/2 (ERK-1/2) was augmented by ES-PAI. Hence, ES-PAI may ameliorate peripheral neuropathy by promoting neuronal cell proliferation and production of neurogenic factors by activating p38 and ERK-1/2 pathways.