Efficacy and safety of denosumab in multiple myeloma.

We analyzed 11 patients with multiple myeloma (MM) treated using denosumab in our institute. The median age was 69 years (range, 54-76 years), and 7 patients were male. Seven patients had presented with newly diagnosed MM. Four patients were initially treated with zoledronic acid, which was then later switched to denosumab. The median number of injections was 15 (range, 1-27). No patients developed skeletal-related events (SRE) during denosumab treatment. Hypocalcemia was defined as serum calcium levels below the baseline in all patients. To prevent hypocalcemia, 8 patients were administered oral calcium. The first course of denosumab injections in 9 patients resulted in more marked decreases in serum calcium concentrations than subsequent courses. Two patients discontinued denosumab after osteonecrosis of the jaw (ONJ) was suspected, but squamous cell carcinoma was later identified in one of these cases. Denosumab might be effective and safe for preventing SRE. Cautionary measurement of serum calcium concentrations and prophylactic supplementation with oral calcium are warranted to avoid hypocalcemia after the first denosumab injection. The possibility of ONJ also merits attention. The clinical diagnosis of ONJ should be considered based on performing appropriate histological analyses.

Differentiation of human skeletal muscle stem cells into odontoblasts is dependent on induction of α1 integrin expression.

Skeletal muscle stem cells represent an abundant source of autologous cells with potential for regenerative medicine that can be directed to differentiate into multiple lineages including osteoblasts and adipocytes. In the current study, we found that α7 integrin-positive human skeletal muscle stem cells (α7(+)hSMSCs) could differentiate into the odontoblast lineage under specific inductive conditions in response to bone morphogenetic protein-4 (BMP-4). Cell aggregates of FACS-harvested α7(+)hSMSCs were treated in suspension with retinoic acid followed by culture on a gelatin scaffold in the presence of BMP-4. Following this protocol, α7(+)hSMSCs were induced to down-regulate myogenic genes (MYOD and α7 integrin) and up-regulate odontogenic markers including dentin sialophosphoprotein, matrix metalloproteinase-20 (enamelysin), dentin sialoprotein, and alkaline phosphatase but not osteoblastic genes (osteopontin and osteocalcin). Following retinoic acid and gelatin scaffold/BMP-4 treatment, there was a coordinated switch in the integrin expression profile that paralleled odontoblastic differentiation where α1ß1 integrin was strongly up-regulated with the attenuation of muscle-specific α7ß1 integrin expression. Interestingly, using siRNA knockdown strategies revealed that the differentiation-related expression of the α1 integrin receptor positively regulates the expression of the odontoblastic markers dentin sialophosphoprotein and matrix metalloproteinase-20. These results strongly suggest that the differentiation of α7(+)hSMSCs along the odontogenic lineage is dependent on the concurrent expression of α1 integrin.

[Bone and Calcium Metabolisms Associated with Dental and Oral-Maxillofacial Diseases. Destructive bone resorption by osteoclasts and oral disease].

Osteoclasts are multinucleated cells that resorb bone, originate from monocyte-macrophage lineage cells. Periodontitis is an inflammatory disease characterized by destruction of periodontal tissues including alveolar bones. Oral implant system is established average dental treatment method. However, peri-implantitis affects the convalescence. Osteonecrosis of the jaw (ONJ) is also inflammatory disease associated with antiresorptive therapy of bisphosphonates. Diagnosis and management of ONJ is more important issue.

Thermoreversible gel for delivery of activin receptor-like kinase 5 inhibitor SB-505124 for glaucoma filtration surgery.

The purpose of this study is to investigate a thermoreversible gel using Pluronic F-127 to deliver an activin receptor-like kinase 5 (ALK-5) inhibitor SB-505124 in glaucoma filtration surgery (GFS). The gel was characterized for in vitro drug release and viscosity studies. Cytotoxicity of Pluronic F-127 was examined by MTT assay using cultured rabbit subconjunctival fibroblasts. In addition, Pluronic F-127 gel (18% w/v) containing 5 mg of SB-505124 was applied at the surgical site in an in vivo rabbit GFS model. In the in vitro viscosity study, the gel showed a change in viscosity (from 1000 cps to 45,000 cps) from low temperature (10°C) to body temperature (37°C). The in vitro drug release study demonstrated 100% drug release within 12 h. The gel did not show cytotoxicity to the cultured rabbit subconjunctival cells by MTT assay. In the in vivo rabbit GFS model, the drug was successfully delivered by injection and no severe post-surgical complications were observed. A thermoreversible gel system with SB-505124 was successfully prepared and delivered for the rabbit GFS model, and it may provide a novel delivery system in GFS.

Matrix metalloproteinase-3 accelerates wound healing following dental pulp injury.

Matrix metalloproteinases (MMPs) are implicated in a wide range of physiological and pathological processes, including morphogenesis, wound healing, angiogenesis, inflammation, and cancer. Angiogenesis is essential for reparative dentin formation during pulp wound healing. The mechanism of angiogenesis, however, still remains unclear. We hypothesized that certain MMPs expressed during pulp wound healing may support recovery processes. To address this issue, a rat pulp injury model was established to investigate expression of MMPs during wound healing. Real-time RT-PCR analysis showed that expression MMP-3 and MMP-9 (albeit lower extent) was up-regulated at 24 and 12 hours after pulp injury, respectively, whereas expression of MMP-2 and MMP-14 was not changed. MMP-3 mRNA and protein were localized in endothelial cells and/or endothelial progenitor cells in injured pulp in vivo. In addition, MMP-3 enhanced proliferation, migration, and survival of human umbilical vein endothelial cells in vitro. Furthermore, the topical application of MMP-3 protein on the rat-injured pulp tissue in vivo induced angiogenesis and reparative dentin formation at significantly higher levels compared with controls at 24 and 72 hours after treatment, respectively. Inhibition of endogenous MMP-3 by N-Isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid resulted in untoward wound healing. These results provide suggestive evidence that MMP-3 released from endothelial cells and/or endothelial progenitor cells in injured pulp plays critical roles in angiogenesis and pulp wound healing.

A novel stem cell source for vasculogenesis in ischemia: subfraction of side population cells from dental pulp.

Cell therapy with stem cells and endothelial progenitor cells (EPCs) to stimulate vasculogenesis as a potential treatment for ischemic disease is an exciting area of research in regenerative medicine. EPCs are present in bone marrow, peripheral blood, and adipose tissue. Autologous EPCs, however, are obtained by invasive biopsy, a potentially painful procedure. An alternative approach is proposed in this investigation. Permanent and deciduous pulp tissue is easily available from teeth after extraction without ethical issues and has potential for clinical use. We isolated a highly vasculogenic subfraction of side population (SP) cells based on CD31 and CD146, from dental pulp. The CD31(-);CD146(-) SP cells, demonstrating CD34+ and vascular endothelial growth factor-2 (VEGFR2)/Flk1+, were similar to EPCs. These cells were distinct from the hematopoietic lineage as CD11b, CD14, and CD45 mRNA were not expressed. They showed high proliferation and migration activities and multilineage differentiation potential including vasculogenic potential. In models of mouse hind limb ischemia, local transplantation of this subfraction of SP cells resulted in successful engraftment and an increase in the blood flow including high density of capillary formation. The transplanted cells were in proximity of the newly formed vasculature and expressed several proangiogenic factors, such as VEGF-A, G-CSF, GM-CSF, and MMP3. Conditioned medium from this subfraction showed the mitogenic and antiapoptotic activity on human umbilical vein endothelial cells. In conclusion, subfraction of SP cells from dental pulp is a new stem cell source for cell-based therapy to stimulate angiogenesis/vasculogenesis during tissue regeneration.

Clinical and radiographic outcomes of posterolateral lumbar spine fusion in humans using recombinant human bone morphogenetic protein-2: an average five-year follow-up study.

The objectives of this study were to determine whether recombinant human bone morphogenetic protein-2 (rhBMP-2) can be used as the sole stimulator of osteogenesis with success equal to an autologous graft in posterolateral lumbar fusion (PLF) at the same level and to describe the progress until bone union. This study included 11 patients who underwent PLF of L4-5. On the right side, only rhBMP-2, for which polylactic/glycolic acid (PLGA) was used as a carrier, was used, whereas, on the left side, autogenous bone was used. The bone union rate was 73 and 82% at 12 and 24 months after surgery, respectively, on the right BMP side, while the rate on the autogenous bone side was 91%. There was no statistically significant difference in the bone union rate. rhBMP-2 can be used as the sole source of osteogenesis with success equivalent to an autologous graft of the PLF.

Immunocytochemical approach for surface layer proteins of freeze-substituted Tannerella forsythensis by energy-filtering transmission electron microscopy.

Tannerella forsythensis (Bacteroides forsythus), an anaerobic gram-negative potential periodontal pathogens in the progression of periodontitis. IT forsythensis has unique bacterial protein profiles containing major proteins with apparent molecular weight of more than 200-kDa shown by sodium dodecylsulfate-polyacrylamide gel electrophoresis. It is also known to have a typical surface layer (S-layer) consisting of regularly arrayed subunits outside the outer membrane revealed by electron microscopy. On the other hand, electron microscopy showed that the best preservation of structure was obtained when cells were postfixed with OsO4, but this resulted in very low levels of gold particles labeling. Therefore, cells were applied to pieces of filter paper and freeze-substituted by plung-freezing in Liquid propane, substituted in methanol containing 0.5% uranyl acetate, and infiltrated with LR-White resin. We also examined the relation between high molecular weight proteins and S-layer in energy-filtering transmission electron microscopy (EF-TEM) to visualize 3,3'-diaminobenzidene, tetrahydrochloride (DAB) reaction. The three-window method in electron spectroscopic images (ESI) of nitrogen (N) element, reflecting the presence of DAB moieties by the DAB reaction solution, horseradish peroxidase (HRP)-conjugated secondary antibodies instead of immunogold particles obtained by the EF-TEM. The mapping patterns of net N element were restricted to the outermost cell surface.

Recovery of Pure Red Cell Aplasia Following Hematopoietic Stem Cell Transplantation Associated with Interleukin (IL)-6 Elevation Caused by Odontogenic Infection.

We herein report a case of long-lasting pure red cell aplasia (PRCA) after major ABO-incompatible allogeneic stem cell transplantation (SCT) for acute lymphoblastic leukemia. The patient needed red blood cell (RBC) transfusion every week after SCT. On day 236, he was diagnosed with odontogenic infection, and the serum levels of Interleukin (IL)-6 were elevated to 12.1 pg/mL. After that, the numbers of reticulocyte rapidly began to increase, and RBC support was not needed from day 251. No standard care for PRCA following SCT has been established. The IL-6 elevation caused by the odontogenic infection therefore appears to have been affected by the improvement in PRCA.

Optically induced melting of colloidal crystals and their recrystallization.

Colloidal crystals melt by applying focused light of optical tweezers and recrystallize after removing it. The disturbed zone by the light grows radially from the focus point and the ordering starts from the interface with the crystal. Although the larger disturbed zone is observed for the higher power optical tweezers, a master curve is extracted by normalization of the disturbed zone. The temporal changes of the normalized disturbed zone are well described with exponential functions, indicating that the melting and recrystallization process is governed by a simple relaxation mechanism.

Photonic band structures of colloidal crystals measured with angle-resolved reflection spectroscopy.

We acquired angle- and polarization-resolved reflection spectra from a colloidal crystal made of polystyrene spheres along the two perpendicular directions corresponding to the LU and LW directions in the first Brillouin zone of an fcc lattice. Dispersion relations between the reflection peak positions and the wave vectors of the incident light were obtained from the measured spectra and compared with calculated photonic band structures. For the first stop band region in the spectra, the behavior of the reflection peak due to Bragg diffraction agreed with the calculated band structure and revealed some differences induced by the polarization and crystalline orientations. The spectral features observed in the higher energy regions also revealed these differences. In addition, dispersion relationships between the peak positions and the wave vectors were obtained from the results of fitting each spectrum with several Gaussian curves, compared with the calculated photonic band structures. The relationships obtained for the LU direction almost matched the calculated band structure, while the relationships obtained for the LW direction revealed the features of the mixed band structure calculated for the two perpendicular directions. These results indicate that angle- and polarization-resolved reflection spectroscopy has the potential to experimentally analyze the photonic band structures of actual photonic crystals.

Development and features of hydro-membrane gas chromatography.

Hydro-membrane gas chromatography (HMGC) is achieved by the annular condensation of water in a capillary column at less than 70 degrees C. The annular membrane of water is formed as a result of the wettability of the stationary phase, which is induced at a water contact angle ranging from 75 degrees to 79 degrees, as derived from a solubility parameter (delta) range of 15.7 +/- 0.3 MPa(1/2) of the coated resin. The range of the liquid to gas volume ratio (beta) required to support the annular membrane should be kept between 0.00005 and 0.0003. In the case of a 0.25-mm i.d. column, the ratio can be set by the combination of a 0.1 to 0.2 microl min(-1) water supply rate and helium gas flow rate. Separation by HMGC develops not only a gas-solid partition but also a focusing effect on the water membrane. One feature of HMGC is that it gives a non-adsorption chromatogram based on the blocking effect of pre-adsorbed water; furthermore, despite the presence of a relatively large quantity of water, the electron impact ionization efficiency is kept the same as in the usual GC/MS condition. The detection limit with the injection of 1 microl of aquatic solution was estimated to be less than 0.1 ppb of low-molecular-weight fatty acids with s/n = 5 on a mass chromatogram at m/z 45. The HMGC/EI-MS system can be applied to the trace analysis of C1 to C3 volatile acids, volatile inorganic acids, and halogenated organic acids in water.

Pulp regeneration by transplantation of dental pulp stem cells in pulpitis: a pilot clinical study.

BACKGROUND: Experiments have previously demonstrated the therapeutic potential of mobilized dental pulp stem cells (MDPSCs) for complete pulp regeneration. The aim of the present pilot clinical study is to assess the safety, potential efficacy, and feasibility of autologous transplantation of MDPSCs in pulpectomized teeth. METHODS: Five patients with irreversible pulpitis were enrolled and monitored for up to 24 weeks following MDPSC transplantation. The MDPSCs were isolated from discarded teeth and expanded based on good manufacturing practice (GMP). The quality of the MDPSCs at passages 9 or 10 was ascertained by karyotype analyses. The MDPSCs were transplanted with granulocyte colony-stimulating factor (G-CSF) in atelocollagen into pulpectomized teeth. RESULTS: The clinical and laboratory evaluations demonstrated no adverse events or toxicity. The electric pulp test (EPT) of the pulp at 4 weeks demonstrated a robust positive response. The signal intensity of magnetic resonance imaging (MRI) of the regenerated tissue in the root canal after 24 weeks was similar to that of normal dental pulp in the untreated control. Finally, cone beam computed tomography demonstrated functional dentin formation in three of the five patients. CONCLUSIONS: Human MDPSCs are safe and efficacious for complete pulp regeneration in humans in this pilot clinical study.

Role of IL-2 and helper T-lymphocytes in limiting periapical pathosis.

The purpose of this study was to examine the role of IL-2 and helper T-lymphocytes in the development of periradicular lesions in rats. In control animals, periradicular lesions developed within 28 days following pulpal infection. Immunologically, some anti IL-2 and anti CD4-reactive helper T-lymphocytes infiltrated the periapical tissue at 14 days, and their numbers increased at 28 days. In experimental animals, tacrolimus (FK506) was injected every day to inhibit the IL-2 production by helper T-cells. Histologically, the pulpal necrosis and periradicular inflammation in tacrolimus-treated rats were more severe than those in the control rats. Furthermore, the areas of pulpal necrosis and periradicular lesion in the immunosuppressed rat were significantly greater than those in the normal ones. The numbers of IL-2- and CD4-positive cells in the lesion of the experimental rats were statistically lower than those of the control ones. These results show that the decrease in IL-2 might have promoted the development of periradicular lesions.

Effectiveness of dental computed tomography in diagnostic imaging of periradicular lesion of each root of a multirooted tooth: a case report.

Radiography by use of the three-dimensional (3D) Accuitomo XYZ Slice View Tomograph (3DX), a device for compact computed tomography in dentistry, was performed as a new diagnostic imaging technique for a patient who needed endodontic therapy. The 3DX was used for examining and diagnosing the presence and expansion of periradicular lesions in each root of a multirooted tooth. High-resolution 3D images were observed and compared with those obtained by routine conventional radiography. It was difficult to diagnose the cause of clinical symptoms by general examinations including the usual intraoral radiography and panoramic radiography. The images obtained by the 3DX, however, clearly showed the apparent presence and expansion of a periradicular lesion in only one root of the multirooted tooth. The 3DX, a newly developed dental computed tomography, provides abundant new image information not attainable by either intraoral radiography or panoramic radiography, and its application for clinical use is very effective for examining and diagnosing regions of interest for endodontic therapy.

Structure-cytotoxicity relationship of methacrylate-based resin monomers as evaluated by an anti-oxidant responsive element-luciferase reporter assay.

To investigate the chemical structure-cytotoxicity relationship of methacrylate-based resin monomers, we studied their effects on anti-oxidant responsive element (ARE)-mediated transcription. HepG2 cells stably expressing an ARE-regulated luciferase reporter gene were cultured for 6 h with various concentrations of several resin monomers and subjected to a luciferase assay. The doseresponse curves observed for hydrophobic monomers with different hydrocarbon chains (MMA, EMA, PMA and BMA) began to rise at concentrations between 0.5 and 1 mM; the curves rose as the monomer concentrations increased up to 5 (BMA), 10 (PMA), or 30 mM (MMA and EMA). In contrast, hydrophilic monomers having a hydroxyl group (HEMA and HPMA) showed bell-shaped curves, and stimulated the reporter expression more strongly than the hydrophobic monomers in a low concentration range (0.5-5 mM). The results suggest that introduction of a hydroxyl group in a methacrylate-based resin monomer increases its intracellular electrophilic reactivity and cytotoxicity.

Inhibitory effect of CGRP on osteoclast formation by mouse bone marrow cells treated with isoproterenol.

The present study was designed to elucidate the mode of action of isoproterenol (Isp; adrenergic beta-agonist) and to characterize the effect of the calcitonin gene-related peptide (CGRP; sensory neuropeptide) on osteoclast formation induced by Isp in a mouse bone marrow culture system. Treatment of mouse bone marrow cells with Isp generated tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells (MNCs) capable of excavating resorptive pits on dentine slices, and caused an increase in receptor activator of NF-kappaB ligand (RANKL) and a decrease in osteoprotegerin (OPG) production by the marrow cells. The osteoclast formation was significantly inhibited by OPG, suggesting the involvement of the RANKL-RANK system. CGRP inhibited the osteoclast formation caused by Isp or soluble RANKL (s-RANKL) but had no influence on RANKL or OPG production by the bone marrow cells treated with Isp, suggesting that CGRP inhibited the osteoclast formation by interfering with the action of RANKL produced by the Isp-treated bone marrow cells without affecting RANKL or OPG production. This in vitro data suggest the physiological interaction of sympathetic and sensory nerves in osteoclastogenesis in vivo.

Evaluation of three methods for effective extraction of DNA from human hair.

In this paper we evaluate three different methods for extracting DNA from human hair i.e. the Chelex method, the QIAamp DNA Mini Kit method and the ISOHAIR method. Analysis of DNA prepared from dyed hairs with the ISOHAIR method suggested that the DNA extracts contained PCR inhibitors. On the other hand, few inhibition was observed when DNA from dyed hairs were extracted using the Chelex method and the QIAamp DNA Mini Kit method. In conclusion, the Chelex method is recommended for PCR experiments in view of its simplicity and cost-effectiveness. To assess the reliability of the Chelex method for the extraction of genomic DNA from both natural and dyed hair samples, minisatellite variant repeat (MVR)-polymerase chain reaction (PCR) patterns of Chelex-extracted DNA were compared using hairs (three natural black hairs and three dyed hairs) with buccal swabs from six individuals. Complete agreement was observed between hair and swab samples in each individual, proving the utility of the Chelex method.