Osteogenic extracellular matrix sheet for bone tissue regeneration.

The application of extracellular matrix (ECM) sheets without a scaffold is not extensively reported in bone regenerative medicine. The aim of the present study was to demonstrate that an osteogenic ECM sheet (OECMS) can retain ECM integrity and growth factors to enhance bone formation in a rat non-union model. OECMS was produced from osteogenic cell sheets (OCS). Collagen and growth factor [bone morphogenetic protein 2 (BMP-2), vascular endothelial growth factors (VFGFs), basic fibroblast growth factor (bFGF) and transforming growth factor ß1 (TGF-ß1)] concentrations in the OECMS were quantified by enzyme-linked immunosorbent assay (ELISA). Next, hydroxyapatite (HA) constructs combined with OECMSs were implanted subcutaneously into the rats' backs to evaluate their osteoinductive capacity by histological evaluation. In addition, OECMSs were implanted in a rat femoral non-union model. 18 male Fischer 344 inbred rats were divided into OECMS and control groups. Fracture healing was evaluated by radiological and histological analyses at 2, 5 and 8 weeks and biological analysis at 8 weeks. Collagen I and growth factors were retained in the OECMSs. Osteoid formation was identified in the HA combined with OECMS at 4 weeks. Enhanced bone regeneration at the non-union of the OECMS group was confirmed at 5 and 8 weeks. Biomechanical testing revealed a significantly higher maximum bending load in the OECMS group as compared to the control group at 8 weeks. The results demonstrated that OECMS retained BMP-2 and TGF-ß1 and high osteoinductive and osteoconductive capacity. As such, OECMS represents a potential new scaffold-free material for bone tissue engineering.

Investigation of the neutron spectrum measurement method for dose evaluation in boron neutron capture therapy.

In boron neutron capture therapy, it is important to evaluate the dose administered to a patient's body outside the tumour area. The exposure dose is evaluated by calculation; however, the calculated value must be validated using a measured value. The dose evaluations based on the measured neutron spectrum are investigated. Multi-foil activation, combined with a LiCaAlF6 scintillation detector and an imaging plate, is proposed as a measurement method. The proposed method can measure the neutron spectrum at various points quickly.

Culturing bone marrow cells with dexamethasone and ascorbic acid improves osteogenic cell sheet structure.

OBJECTIVES: To assess the structure and extracellular matrix molecule expression of osteogenic cell sheets created via culture in medium with both dexamethasone (Dex) and ascorbic acid phosphate (AscP) compared either Dex or AscP alone. METHODS: Osteogenic cell sheets were prepared by culturing rat bone marrow stromal cells in a minimal essential medium (MEM), MEM with AscP, MEM with Dex, and MEM with Dex and AscP (Dex/AscP). The cell number and messenger (m)RNA expression were assessed in vitro, and the appearance of the cell sheets was observed after mechanical retrieval using a scraper. ß-tricalcium phosphate (ß-TCP) was then wrapped with the cell sheets from the four different groups and subcutaneously implanted into rats. RESULTS: After mechanical retrieval, the osteogenic cell sheets from the MEM, MEM with AscP, and MEM with Dex groups appeared to be fragmented or incomplete structures. The cell sheets cultured with Dex/AscP remained intact after mechanical retrieval, without any identifiable tears. Culture with Dex/AscP increased the mRNA and protein expression of extracellular matrix proteins and cell number compared with those of the other three groups. More bridging bone formation was observed after transplantation of the ß-TCP scaffold wrapped with cell sheets cultured with Dex/AscP, than in the other groups. CONCLUSIONS: These results suggest that culture with Dex/AscP improves the mechanical integrity of the osteogenic cell sheets, allowing retrieval of the confluent cells in a single cell sheet structure. This method may be beneficial when applied in cases of difficult tissue reconstruction, such as nonunion, bone defects, and osteonecrosis.Cite this article: M. Akahane, T. Shimizu, T. Kira, T. Onishi, Y. Uchihara, T. Imamura, Y. Tanaka. Culturing bone marrow cells with dexamethasone and ascorbic acid improves osteogenic cell sheet structure. Bone Joint Res 2016;5:569-576. DOI: 10.1302/2046-3758.511.BJR-2016-0013.R1.

A new ultrasound-guided pubic approach for proximal obturator nerve block: clinical study and cadaver evaluation.

We evaluated an alternative technique for ultrasound-guided proximal level obturator nerve block that might facilitate needle visualisation using in-plane ultrasound guidance. Twenty patients undergoing transurethral bladder tumour resection requiring an obturator nerve block were enrolled into a prospective observational study. With the patient in the lithotomy position, the transducer was placed on the medial thigh along the extended line of the inguinal crease, and aimed cephalad to view a thick fascia between the pectineus and obturator externus muscles that contains the obturator nerve. A stimulating nerve block needle was inserted at the pubic region and advanced in-plane with the transducer in an anterior-to-posterior direction. Eight ml levobupivacaine 0.75% was injected within the fascia. The median (IQR [range]) duration for ultrasound identification of the target and injection were 8.5 (7-12 [5-24]) s and 62 (44.5-78.25 [39-383]) s, respectively. All blocks were successful. A cadaver evaluation demonstrated that the dye injected into the target fascia using our technique travelled retrogradely through the obturator canal, and surrounded the anterior and posterior branches of the obturator nerve both proximally and distally to the obturator canal. We believe that this is a promising new technique for ultrasound-guided proximal level obturator nerve block.

Nadir PSA level and time to PSA nadir following primary androgen deprivation therapy are the early survival predictors for prostate cancer patients with bone metastasis.

The purpose of this study was to identify the early predictor of survival for prostate cancer patients with bone metastasis. We reviewed 87 prostate cancer patients with bone metastasis who had received primary androgen deprivation therapy (PADT) at our institution. The medical records of the patients were examined with respect to laboratory data, pathological results, PSA response to PADT and clinical outcome. The overall survival (OS) rates were analyzed with reference to the nadir PSA level and time to PSA nadir (TTN) following PADT by Kaplan-Meier method. In all, 59 patients (67%) had progression to castration-resistant prostate cancer. Nadir PSA <0.2 ng ml(-1) (lower PSA nadir) during PADT were observed in 47 patients (54%). Multivariate analysis revealed that the extent of disease on bone scan (P=0.04), lower PSA nadir following PADT (P=0.003), albumin (P=0.04) and lactate dehydrogenase (P=0.01) were independent prognostic factors for survival. OS rates in the patients with lower PSA nadir were significantly higher. Longer TTN (>9 months) identified patients with prolonged OS in both lower and higher PSA nadir groups. PSA nadir <0.2 ng ml(-1) and prolonged TTN (>9 months) following PADT might be the most important early predictors for longer survival in prostate cancer patients with bone metastasis.

Non-sentinel lymph node status and prognosis of breast cancer patients with micrometastatic sentinel lymph nodes.

BACKGROUND: The prognostic significance of sentinel lymph node (SLN) micrometastases and the need for axillary lymph node dissection (ALND) on patients with micrometastases in SLNs remain controversial. METHODS: A prospective database of 657 breast cancer patients who underwent SLN biopsy (SLNB) was analyzed. SLNs were detected using a combined method of isosulfan blue dye and small-sized technetium-99m-labeled tin colloid. RESULTS: Micrometastases in SLNs were found in 50 (7.6%) of 657 patients. Twenty-nine (58.0%) of 50 patients with micrometastatic SLNs underwent ALND and no further metastases were found in non-sentinel lymph nodes. Among 21 patients (42.0%) with micrometastatic SLNs who decided to forego ALND, no axillary lymph node recurrence has been observed during a median follow-up time of 47 months. There is no significant difference in recurrence-free survival between the patients with micrometastatic and negative SLNs (p = 0.90). CONCLUSIONS: These data suggest that it may not be necessary to perform ALND on patients with micrometastases in SLNs and that the presence of micrometastases in SLNs may not be associated with prognosis.

Superiority of radioisotope over blue dye for sentinel lymph node detection in breast cancer.

BACKGROUND: Sentinel lymph node biopsy (SLNB) is commonly performed using radioisotopes and/or blue dye. However, it is still undefined which reagent is more suitable for identifying sentinel lymph nodes (SLN). PATIENTS AND METHODS: A consecutive series of 640 breast cancer patients who had undergone SLNB at the Keio University Hospital from 2001 to 2006 was analyzed. The SLN was identified by a combination of technetium-99m tin colloid and isosulfan blue dye. The correlation between clinicopathological factors and the distribution of radioisotopes and blue dye was analyzed. The single metastatic lymph node revealed by axillary lymph node dissection (ALND) is the 'true SLN', and the distribution of radioisotopes and blue dye to the 'true SLN' was also analyzed. RESULTS: Blue-dye- and radioisotope-positive SLN were identified in 79.6 and 94.7% of the patients, respectively. Taken together, SLN were identified in 625 patients (97.7%) by radioisotope and/or blue dye. No significant correlation was observed between clinicopathological features and the distribution of the reagents. ALND found 73 patients with single lymph node metastasis, and 73 'true SLN' were identified by blue dye in 65.7% (48/73), and by radioisotope in 95.9% (70/73) of the cases. CONCLUSION: These data suggest that radioisotopes are superior to blue dye in detecting SLN in breast cancer.

An aspergillotic aneurysm of the internal carotid artery following allogeneic bone marrow transplantation: successful management with catheter coil embolization and long-term antifungal agents.

We report a case of a mycotic aneurysm of the internal carotid artery and cerebral hemorrhagic infarction resulting from Aspergillus middle ear infection in a patient with severe aplastic anemia who received unrelated bone marrow transplantation. Although a mycotic aneurysm is a rare complication, and most often fatal, the patient was successfully treated with catheter coil embolization of the internal carotid artery and long-term systemic antifungal therapy. This case emphasizes the need for the rapid diagnosis of potential fungal involvement of the vascular system and suggests the necessity for aggressive treatment, such as with the modality illustrated in this case.

Phex mutation causes overexpression of FGF23 in teeth.

OBJECTIVE: Hyp mice have a disorder in phosphate homeostasis, and display hypo-mineralization in bones and teeth, while the Phex (phosphate regulating gene homologies to endopeptidase on the X chromosome) gene in Hyp mice has a deletion of the 3' end. We investigated whether a mutation of Phex has an effect on the expression level of fibroblast growth factor 23 (FGF23), one of the key factors of phosphate homeostasis, in developing teeth of Hyp mice. DESIGN: RT-PCR and in situ hybridisation analyses for FGF23 were performed using developing teeth of WT mice. Quantitative RT-PCR analyses for FGF23 were performed using the tooth germs of WT and Hyp mice in both in vivo and in vitro experiments. RESULTS: Undifferentiated and early secretory ameloblasts as well as odontoblasts expressed FGF23 mRNA during early tooth development. Further, quantitative RT-PCR analyses revealed that the amount of FGF23 mRNA in Hyp mouse teeth was significantly higher than that in wild type mice. CONCLUSIONS: These findings suggest that loss of Phex function is related to overexpression of FGF23 in teeth, which is an intrinsic defect of Hyp mouse teeth.

Remineralization effects of gum arabic on caries-like enamel lesions.

OBJECTIVE: Gum arabic is a natural polysaccharide exudate from Acacia senegal and other related African species of Acacia. Gum arabic is considered to have an ability to enhance remineralization, because of its high concentration of Ca(2+). However, the caries preventive capacity of gum arabic has been scarcely investigated. We evaluated the cariostatic activities of gum arabic using histopathological methods to determine its effects on remineralization. DESIGN: Following incubation in demineralization solution, human third molars were exposed to 10 mg/ml of gum arabic, sodium fluoride at 1000 ppm (NaF), or double distilled water (DW, negative control), then subjected to demineralization-remineralization cycles. Before and after demineralization-remineralization cycles, contact microradiographs of each sample were taken and mineral distribution quantities were calculated. RESULTS: The remineralization ratio of the molars exposed to gum arabic was similar to that of those exposed to NaF, while the ratios of both were significantly greater than that of those exposed to DW. CONCLUSIONS: Gum arabic enhanced the remineralization of caries-like enamel lesions in vitro, suggesting its inhibitory effects towards dental caries.

Phex mutation causes the reduction of npt2b mRNA in teeth.

Hyp mice (murine homologue of human X-linked hypophosphatemia) have a disorder in phosphate homeostasis, and display hypomineralization in bones and teeth. We investigated whether a mutation of Phex (phosphate regulating gene homologies to endopeptidase on the X chromosome) has an effect on the expression level of type II sodium-dependent phosphate co-transporter (Npt2) in the developing teeth of the Hyp mouse. Quantitative RT-PCR analyses revealed that the amount of Npt2b mRNA, an isoform of Npt2, in Hyp mouse tooth germs was significantly lower than that in wild-type mice, in both in vivo and in vitro experiments. In addition, tooth germs from wild-type mice cultured in medium supplemented with antisense oligo-deoxynucleotide for Phex also showed a reduction of Npt2b mRNA expression. These findings suggest that the loss of Phex function is related to the defect of Npt2b expression in teeth, and Npt2b reduction is an intrinsic defect of Hyp murine teeth.

Clinical characteristics of fulminant-type autoimmune hepatitis: an analysis of eleven cases.

BACKGROUND: Although a few adult cases of fulminant-type autoimmune hepatitis have been reported, their clinical features and prognosis have remained uncertain. AIM: To assess the clinical features and prognosis of patients with fulminant-type autoimmune hepatitis. METHODS: Eleven patients (10%) diagnosed with fulminant-type autoimmune hepatitis in accordance with the 1999 criteria of the International Autoimmune Hepatitis Group were analysed. RESULTS: All 11 patients were female, with a median age of 53 years. Five patients survived without liver transplantation, one received a liver transplantation, and five died without liver transplantation. Nine patients (82%) survived for 2 weeks or more following diagnosis, without liver transplantation. Except for the patient receiving a liver transplantation, serum total bilirubin levels measured during the clinical course were significantly higher in non-survivors than in survivors, although the accompanying serum alanine aminotransferase levels measured for the two groups were similar. Most significantly, serum total bilirubin levels in non-survivors worsened during days 8-15, while levels in survivors improved during the same period. CONCLUSIONS: The short-term prognosis for patients with fulminant-type autoimmune hepatitis may be good. However, patients whose serum total bilirubin levels worsen during days 8-15 should be considered for liver transplantation.

Peripheral blood stem cell mobilization by granulocyte colony-stimulating factor alone and engraftment kinetics following autologous transplantation in children and adolescents with solid tumor.

In 56 pediatric and adolescent patients (median age 7 years, range 1-21) with various solid tumors, peripheral blood stem cells (PBSC) were mobilized with granulocyte colony-stimulating factor (G-CSF) alone, and the yields of PBSC and engraftment kinetics following autologous peripheral blood stem cell transplantation (PBSCT) were evaluated retrospectively. Granulocyte colony-stimulating factor (10 microg/kg) was injected subcutaneously for mobilization when patients showed no influence of previous chemotherapy, and administration was continued for 5 days. The peaks of CD34+ cells and colony-forming units-granulocyte/macrophage in the blood were observed on days 4 through 6 of G-CSF administration in all patients. Peripheral blood stem cell harvest was commenced on day 5 of G-CSF treatment. Compared to the results in patients mobilized by chemotherapy plus G-CSF (N=18), the progenitor cell yields were lower in patients mobilized with G-CSF alone. However, there were no significant differences in WBC and ANC engraftment compared to the chemotherapy plus G-CSF mobilization group. Platelet recovery following autologous PBSCT was delayed in patients mobilized with G-CSF alone. The median time taken for ANC and platelet counts to reach 0.5 x 10(9) and 20 x 10(9)/l was 12 days (range: 9-28) and 15 days (8-55), respectively, in all patients who received PBSC mobilized by G-CSF alone. In summary, mobilization with G-CSF alone can mobilize sufficient CD34+ cells for successful autografting and sustained hematological reconstitution in pediatric and adolescent patients with solid tumors, and even in heavily pre-treated patients.

Dentinal defects in Hyp mice not caused by hypophosphatemia alone.

The Hyp mouse is a murine homolog of human X-linked hypophosphatemic rickets and displays hypo-mineralization in bone and dentin due to a defect of the phosphate-regulating gene with homology to endopeptidase on the X chromosome (Phex) gene. It has long been considered that the bone and dentin defects in Hyp mice are caused by hypophosphatemia alone, however, several recent studies have indicated the possibility that intrinsic defects are present in Hyp mice osteoblasts. Further, we previously found a hyper-expression of osteocalcin (OC) mRNA in Hyp mouse odontoblasts and suggested the possibility of the presence of intrinsic defects. In the present study, we evaluated morphological features and OC mRNA expression levels in tooth germs of Nor mice with a normal phex gene and a low concentration of serum phosphate, and compared them to those in Hyp and wild-type mice. Nor mice exhibited low serum phosphate levels, however, did not show the characteristic features of dentin defects seen in Hyp mice, such as widened predentin and hyper-expression of OC mRNA. These results suggest that the hypo-mineralization of dentin in Hyp mice is not dependent on serum phosphate level, but rather is affected by intrinsic defects in odontoblasts.

Factors associated with granulocyte colony-stimulating factor-induced peripheral blood stem cell yield in healthy donors.

BACKGROUND AND OBJECTIVES: Poor collection results are a clinical problem in granulocyte-colony stimulating factor (G-CSF)-induced peripheral blood stem cell (PBSC) collection in healthy donors. It would be beneficial to be able to predict the PBSC yield from allogeneic donors before mobilization or harvesting. MATERIALS AND METHODS: We examined the relationship between certain donor characteristics and the effectiveness of G-CSF-induced PBSC collection in 59 healthy family donors aged 3-63 years old (median 16 years). G-CSF was administered subcutaneously at 10 microg/kg for mobilization, daily for 5 days, and PBSC harvest using a continuous blood cell separator was started on day 5 of G-CSF treatment. Total cell yields were calculated as the number per unit of processed blood (l) per unit weight of the donor (kg). RESULTS: In a univariate analysis, the donor's age, body mass index (BMI), white blood cell (WBC) count before mobilization, and platelet count before and during mobilization were significantly correlated with the yield of mononuclear cells (MNC), CD34(+) cells and granulocyte-macrophage colony-forming units (GM-CFU). Younger age (P < 0.001), a low BMI (P = 0.002), a high WBC count before mobilization (P = 0.004), a high platelet count before (P = 0.012) and during (P < 0.05) mobilization, and a low speed of withdrawal (P = 0.019) were associated with a higher CD34(+) cell yield. No significant correlation was found for gender, the type of G-CSF, the serum level of G-CSF, the type of cell separator, or the type of blood access. A multivariate forward and backward stepwise selection regression analysis showed that the factors associated with CD34(+) cell yield were age, platelet count before and during mobilization, and circulating CD34(+) cell concentration on day 2 of G-CSF treatment. CONCLUSION: In this small preliminary study, we found that donor age is the most important factor in predicting G-CSF-induced PBSC yields. Old age and low platelet counts before mobilization might be useful indicators for identifying poor mobilizers. Further validation of these findings in a larger number of donors are needed to establish whether these findings apply to other populations.

Hyper-expression of osteocalcin mRNA in odontoblasts of Hyp mice.

The Hyp mouse is a murine homologue of human X-linked hypophosphatemia that displays hypo-mineralization in bone and dentin. In this study, we tested the hypothesis that the defect in Hyp mice leads to alterations in the expression of dentin matrix proteins that may be associated with the hypo-mineralization changes in the tissues. Quantitative RT-PCR analyses showed that expression of the osteocalcin gene in Hyp mice tooth germ samples was significantly higher than in wild-type mice, whereas the gene expressions of osteonectin, osteopontin, dentin matrix protein 1, and type I collagen in both types of mice were similar. Further, cultured Hyp mice tooth germ samples exhibited a higher expression of the osteocalcin gene than did those from wild-type mice, which was in accord with the results of our in vivo analysis. These findings suggest that osteocalcin mRNA is highly expressed in Hyp mice odontoblasts and may be associated with dentin hypo-mineralization.

Anomalously hindered E2 strength B(E2;2+(1)-->0+) in 16C.

The electric quadrupole transition from the first 2(+) state to the ground 0(+) state in 16C is studied through measurement of the lifetime by a recoil shadow method applied to inelastically scattered radioactive 16C nuclei. The measured mean lifetime is 77+/-14(stat)+/-19(syst) ps. The central value of mean lifetime corresponds to a B(E2;2+(1)-->0(+)) value of 0.63e(2) fm(4), or 0.26 Weisskopf units. The transition strength is found to be anomalously small compared to the empirically predicted value.

Semiclassical study on tunneling processes via complex-domain chaos.

We investigate the semiclassical mechanism of tunneling processes in nonintegrable systems. The significant role of complex-phase-space chaos in the description of the tunneling processes is elucidated by studying a kicked scattering model. Behaviors of tunneling orbits are encoded into symbolic sequences based on the structure of a complex homoclinic tangle. By means of the symbolic coding, the phase space itineraries of tunneling orbits are related with the amounts of imaginary parts of actions gained by the orbits, so that the systematic search of dominant tunneling orbits becomes possible.