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.

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.

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.

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.

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.

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.

Effects of extracts of rat tissues on the action of bleomycin.

The cell sap (105 000 times g supernatant) of various tissues of rats caused DNA degradation in the presence of bleomycin. The activity was fractionated into two peaks by column chromatography on Sephadex G-25. The activity in Peak A (excluded fraction) appeared to be due to some proteinaceous entity, while that recovered in Peak B (retarded fraction), constituting about 90% of the total activity, seemed to be due to ascorbic acid, judging by results of further gel filtration and the effect of treatment with ascorbate oxidase. Incubation of bleomycin with Peak A or B caused loss of the ability of the antibiotic to degrade DNA. It is proposed that the action of bleomycin on DNA, and its inactivation by tissue extracts, depend, at least in part, on the presence of ascorbic acid.

Thiamin uptake in Euglena gracilis.

Thiamin uptake has been investigated in Euglena gracilis Z. This protozoon possessed an active transport system for thiamin with a Km value of 17 nM and a Vmax value of 7.8 pmol per 10(6) cells per min. Thiamin uptake was dependent on pH and temperature, but not on exogenous glucose as an energy source. Oxythiamin and pyrithiamin were competitive inhibitors with Ki values of 33 nM and 15 nM, respectively. Thiamin monophosphate, thiamin pyrophosphate, thiamin triphosphate, heteropyrithiamin, quinolinothiamin, thiamin chloride and amprolium inhibited uptake. Inhibition of thiamin uptake by various metabolic inhibitors and anaerobiosis suggest that thiamin uptake requires an energy source generated by respiration and glycolysis.

Mitogen-activated protein kinase pathway is involved in alpha6 integrin gene expression in androgen-independent prostate cancer cells: role of proximal Sp1 consensus sequence.

Metastatic diseases of prostate cancer reveal high expression of alpha6 integrin and the activation of mitogen-activated protein kinases (MAP kinase). Therefore, the present study was conducted to examine whether MAP kinase pathway is involved in the alpha6 integrin gene expression in androgen-independent prostate cancer cell lines. alpha6 integrin mRNA expression, the alpha6 integrin promoter-induced luciferase activities and MAP kinase enzyme activities in androgen-independent LNCaP and PC-3 cell lines were higher than those in androgen-dependent LNCaP. Deletion and mutation analysis showed that Sp1 consensus sequence at -48 to -43 bp from the transcription start site was necessary for basal promoter activity. Binding of Sp1 to its consensus sequence in three cell lines was confirmed by electrophoretic mobility shift assays. Sp1 binding to its consensus sequence, as well as promoter activity and mRNA expression, were found to be inhibited by an inhibitor of MAP kinase kinase 1 and 2, U0126, in the androgen-independent cell lines. Our results indicate that the proximal Sp1 is necessary for basal promoter activity of the alpha6 integrin, suggesting that signal transduction from MAP kinases to activation of Sp1 might be involved in alpha6 integrin gene expression in androgen-independent prostate cancer cell lines.

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.

The mitogenic effect of parathyroid hormone is associated with E2F-dependent activation of cyclin-dependent kinase 1 (cdc2) in osteoblast precursors.

Injections of parathyroid hormone (PTH) have been reported to stimulate skeletal accretion through increased bone formation in several species, and osteoblast proliferation is a critical component of bone formation. However, the biological mechanisms of PTH-stimulated bone cell proliferation are largely unknown. In this study, we demonstrated that PTH stimulates proliferation of the osteoblast precursor cell line, TE-85, in association with increasing cdc2 protein levels and its kinase activity. cdc2 antisense oligonucleotides blocked PTH-induced DNA synthesis and cell cycle progression. Analysis of the time course of PTH-stimulated cdc2 message levels demonstrated that cdc2 mRNA levels were increased 1.5- to 4-fold between 3-18 h following release from cell synchronization. Transfections of TE-85 cells with a series of cdc2 promoter-luciferase deletion constructs revealed PTH stimulation of the cdc2 promoter. Promoter constructs containing a mutation in the E2F binding site were not stimulated by PTH. Gel mobility shift assays demonstrated increased free E2F levels in TE-85 nuclear extracts in response to PTH. Furthermore, the ratios of hyperphosphorylated to hypophosphorylated forms of Rb protein were increased by PTH treatment. These results demonstrate that PTH-stimulated cdc2 expression was associated with TE-85 cell proliferation and that the mechanism of stimulating cdc2 gene expression involved increasing the levels of free E2F.

Localization of Smads, the TGF-beta family intracellular signaling components during endochondral ossification.

Members of the transforming growth factor-beta (TGF-beta) family transduce signals from the cell membrane to the nucleus via specific type I and type II receptors and Smad proteins. Smad1 and Smad5 mediate intracellular signaling of bone morphogenetic protein (BMP), whereas Smad2 and Smad3 transduce TGF-beta signaling. Smad4 is a common mediator required for both pathways. Smad6 and Smad7 inhibit signaling by members of the TGF-beta superfamily. Here, we examined the expression of Smad1 to Smad7 proteins during endochondral ossification of epiphyseal plate of growing rats using immunohistochemical techniques. The expression of Smad proteins was correlated with the expression of TGF-beta1 and its receptors, and BMP-2/4 and BMP receptors. The results show that TGF-beta1 and BMP-2/4 were actively expressed in chondrocytes that are undergoing proliferation and maturation, which overlaps with expression of their corresponding type I and type II receptors. The Smads, however, exhibited a distinct expression pattern, respectively. For example, Smad1 and Smad5 were highly expressed in proliferating chondrocytes and in those chondrocytes that are undergoing maturation. The TGF-beta/activin-restricted Smads were also expressed in a nearly complementary fashion; Smad2 was strongly expressed in proliferating chondrocytes, whereas Smad3 was strongly observed in maturing chondrocytes. Smad4 was broadly expressed in all zones of epiphyseal plate. Inhibitory Smads, Smad6 and Smad7, were strongly expressed in the zone of cartilage that contained mature chondrocytes. Our findings show a colocalization of the pathway-restricted and inhibitory Smads with activating ligands or ligands whose action they antagonize and their receptors in various zones of epiphyseal growth plate, suggesting that TGF-beta superfamily Smad signaling pathways plays a morphogenic role during endochondral bone formation.

Association of increased intracellular free Ca2+ by platelet-derived growth factor with mitogenesis but not with proteoglycan synthesis in chondrocytes--effect of suramin.

The effects of platelet-derived growth factor (PDGF) on the intracellular free Ca2+ concentration [( Ca2+]i) in chondrocytes were studied with a fluorescent Ca2+ indicator, fura 2, and compared with the effects of PDGF on mitogenesis and proteoglycan synthesis. PDGF evoked phasic and then tonic increase in [Ca2+]i dose-dependently in quiescent cultures of chondrocytes, and it also stimulated both DNA and proteoglycan syntheses dose-dependently similar to somatomedins. Suramin, which inhibits the interaction of PDGF with its receptors, caused dose-dependent inhibition of both the PDGF-evoked increase in [Ca2+]i and stimulation of DNA synthesis by PDGF. However, suramin rather enhanced the proteoglycan synthesis induced by PDGF without affecting the basal level of proteoglycan synthesis directly. These results suggest that [Ca2+]i may be an important signal for the action of PDGF on cell proliferation in chondrocytes, and that the initial signal for proteoglycan synthesis is different from that for DNA synthesis induced by PDGF after the activation of PDGF receptor.