Self-Adherent Biodegradable Gelatin-Based Hydrogel Electrodes for Electrocardiography Monitoring.

Patch-type hydrogel electrodes have received increasing attention in biomedical applications due to their high biocompatibility and conformal adherence. However, their poor mechanical properties and non-uniform electrical performance in a large area of the hydrogel electrode should be improved for use in wearable devices for biosignal monitoring. Here, we developed self-adherent, biocompatible hydrogel electrodes composed of biodegradable gelatin and conductive polymers for electrocardiography (ECG) measurement. After incorporating conductive poly(3,4-ethylenedioxythiophene):poly(4-styrenesulfonate) (PEDOT:PSS) into gelatin hydrogels crosslinked by natural crosslinkers (genipin), the mechanical properties and electrical conductivity of the hydrogel electrodes were improved and additionally optimized by adjusting the amounts of crosslinker and PEDOT:PSS, respectively. Furthermore, the effect of dimethyl sulfoxide, as a dopant, on the conductivity of hydrogels was investigated. The gelatin-based, conductive hydrogel patch displayed self-adherence to human skin with an adhesive strength of 0.85 N and achieved conformal contact with less skin irritation compared to conventional electrodes with a chemical adhesive layer. Eyelet-type hydrogel electrodes, which were compatible with conventional ECG measurement instruments, exhibited a comparable performance in 12-lead human ECG measurement with commercial ECG clinical electrodes (3M Red Dot). These self-adherent, biocompatible, gelatin-based hydrogel electrodes could be used for monitoring various biosignals, such as in electromyography and electroencephalography.

Metal Ion-Mediated Interliposomal Aggregation of Polydiacetylene Liposomes Incorporating a Phenolic Lipid.

Intercolloidal behaviors mediated by metal-ligand coordination have rarely been studied. In this work, such intercolloidal behaviors were demonstrated visibly using blue-colored polydiacetylene liposomes containing a phenolic lipid that acts as a binding ligand toward metal ions. The optimized liposomes were 150-200 nm in diameter and stable in aqueous solution. In incubation tests with various neocortical metal ions, iron(III) ions produced the most obvious colloidal aggregation of the liposomes. As the pH of the incubation medium was increased from acid to basic, stronger aggregation and increased precipitation behavior were observed. The phenolic lipid is believed to contribute to the interliposomal bridging interaction, and the pH dependence of the complexation between iron(III) and the phenolic lipid inserted in the liposomes were verified.

Sendai viroplexes for epidermal growth factor receptor-directed delivery of interleukin-12 and salmosin genes to cancer cells.

BACKGROUND: The effective delivery of therapeutic genes to target cells has been a fundamental goal in cancer gene therapy because of its advantages with respect to both safety and transfection efficiency. In the present, study we describe a tumor-directed gene delivery system that demonstrates remarkable efficacy in gene delivery and minimizes the off-target effects of gene transfection. METHODS: The system consists of a well-verified cationic O,O'-dimyristyl-N-lysyl glutamate (DMKE), Sendai virus fusion (F) protein and hemagglutinin-neuraminidase (HN) protein, referred to as cationic Sendai F/HN virosomes. To achieve tumor-specific recognition, anti-epidermal growth factor (EGF) receptor antibody was coupled to the surface of the virosomes containing interleukin-12 (IL-12) and/or salmosin genes that have potent anti-angiogenetic functions. RESULTS: Among the virosomal formulations, the anti-EGF receptor (EGFR) viroplexes, prepared via complexation of plasmid DNA (pDNA) with cationic DMKE lipid, exhibited more efficient gene transfection to tumor cells over-expressing EGF receptors compared to the neutrally-charged anti-EGFR virosomes encapsulating pDNA. In addition, the anti-EGFR viroplexes with IL-12 and salmosin genes exhibited the most effective therapeutic efficacy in a mouse tumor model. Especially when combined with doxorubicin, transfection of the two genes via the anti-EGFR viroplexes exhibited an enhanced inhibitory effect on tumor growth and metastasis in lungs. CONCLUSIONS: The results of the present study suggest that anti-EGFR viroplexes can be utilized as an effective strategy for tumor-directed gene delivery. Copyright © 2016 John Wiley & Sons, Ltd.

Hepatic control elements promote long-term expression of human coagulation factor IX gene in hydrodynamically transfected mice.

BACKGROUND: Long-term expression of the delivered target gene is critical for successful gene therapy. Recently, hepatic control region I (HCR I) originating from the apolipoprotein (apo)C-I pseudogene was shown to be a critical element for long-term gene expression in the liver of mice. HCR II is another hepatic control region of apoC-I. METHODS: HCR I, HCR II and HCR I/II-containing plasmids encoding factor IX were prepared and hydrodynamically transferred into the liver of normal and hemophilia B mice. Factor IX expression, clotting activity and formation of antibodies against the expressed gene product were compared. RESULTS: HCR I-, HCR II- and HCR I/II-containing plasmids all induced long-term gene expression in both normal and hemophilia B mice. Post-transfection factor IX expression in the hemophilia B mice remained above 500 ng/ml for 210 days. Antibodies against human factor IX were detected at a low level in the serum, although they had no effect on the levels and clotting activity of the expressed factor IX. CONCLUSIONS: We have shown in mouse models that hydrodynamic transfection of pBS-HCRII-HP-FIXA and pBS-HCRI/II-HP-FIXA was able to induce and maintain the expression and clotting activity of human factor IX for a long period of time at a potentially therapeutic level. With an appropriate delivery system, this type of plasmid vector could be clinically useful for the hepatic expression of therapeutic genes including human factor IX.

Intrarectal vaccination with recombinant vaccinia virus expressing carcinoembronic antigen induces mucosal and systemic immunity and prevents progression of colorectal cancer.

The gastrointestinal mucosa contains an intact immune system that protects the host from pathogens and communicates with the systemic immune system. Absorptive epithelial cells in the mucosa give rise to malignant tumors although the interaction between tumor cells and the mucosal immune system is not well defined. The pathophysiology of colorectal cancer has been elucidated through studies of hereditary syndromes, such as familial adenomatous polyposis, a cancer predisposition syndrome caused by germline mutations in the adenomatous polyposis coli tumor suppressor gene. Patients with FAP develop adenomas and inevitably progress to invasive carcinomas by the age of 40. To better delineate the role of mucosal immunity in colorectal cancer, we evaluated the efficacy of intrarectal recombinant vaccinia virus expressing the human carcinoembryonic Ag (CEA) in a murine FAP model in which mice are predisposed to colorectal cancer and also express human CEA in the gut. Mucosal vaccination reduced the incidence of spontaneous adenomas and completely prevented progression to invasive carcinoma. The therapeutic effects were associated with induction of mucosal CEA-specific IgA Ab titers and CD8(+) CTLs. Mucosal vaccination was also associated with an increase in systemic CEA-specific IgG Ab titers, CD4(+) and CD8(+) T cell responses and resulted in growth inhibition of s.c. implanted CEA-expressing tumors suggesting communication between mucosal and systemic immune compartments. Thus, intrarectal vaccination induces mucosal and systemic antitumor immunity and prevents progression of spontaneous colorectal cancer. These results have implications for the prevention of colorectal cancer in high-risk individuals.

Local IL-21 promotes the therapeutic activity of effector T cells by decreasing regulatory T cells within the tumor microenvironment.

The eradication of tumors by the immune system depends on the generation of antigen-specific T cells which can migrate to sites of tumor growth and maintain their effector functions despite local tumor-derived T-cell inhibitory factors. Interleukin-21 (IL-21) is an IL-2-related cytokine that has shown limited evidence of antitumor activity in murine models and early phase clinical trials. Effect of local IL-21 on T-cell responses within the tumor microenvironment, however, has not been extensively evaluated. Thus, we developed a stably transfected IL-21-secreting B16 melanoma cell line to test the effects of local IL-21 on endogenous and adoptively transferred T-cell responses. Tumors expressing IL-21 exhibited delayed growth in vivo, which was associated with an increase in activated systemic effector and memory CD8(+) T-cell responses. Local IL-21 also enhanced the therapeutic effects of adoptively transferred gp100-specific T cells and was synergistic with IL-2. The effect was also associated with an increased proliferation of local CD8(+) T cells and decreased accumulation of regulatory CD4(+)FOXP3(+) T cells within the tumor microenvironment. These data suggest that local IL-21 enhances endogenous and adoptively transferred T-cell immunity through increased effector CD8(+) T cells and decreased CD4(+) regulatory T cells in the tumor microenvironment.

Tumor-specific delivery of therapeutic siRNAs by anti-EGFR immunonanoparticles.

BACKGROUND: Efficient target-specific siRNA delivery has always been a primary concern in the field of siRNA clinical application. PURPOSE: In this study, four different types of anti-epidermal growth factor receptor (EGFR) antibody-conjugated immunonanoparticles were prepared and tested for cancer cell-targeted therapeutic siRNA delivery. MATERIALS AND METHODS: The prepared nanoparticles encapsulating siRNAs were character-ized by gel retardation and particle analysis using a Zetasizer. In vitro transfection and reduction of target genes, vimentin and JAK3, were determined using quantitative reverse transcription polymerase chain reaction. In vivo tumor targeting and antitumoral efficacies of the nanoparticles were evaluated in mice carrying tumors. RESULTS: Among these immunonanoparticles, anti-EGFR immunolipoplexes and immunoviroplexes exhibited remarkable cell binding and siRNA delivery to EGFR-expressing tumor cells compared to immunoliposomes and immunovirosomes. Especially, the anti-EGFR immunoviroplexes exhibited the most efficient siRNA transfection to target tumor cells. Therefore, antitumoral vimentin and Janus kinase-3 siRNAs were loaded in the anti-EGFR immunolipoplexes and immunoviroplexes, which were tested in mice carrying SK-OV-3 tumor xenografts. In fact, the therapeutic siRNAs were efficiently delivered to the tumor tissues by both delivery vehicles, resulting in significant inhibition of tumor growth. Moreover, administration of doxorubicin in combination with anti-EGFR immunoviroplexes resulted in remarkable and synergistic tumor growth inhibition. CONCLUSION: This study provides experimental proof that cancer cell-targeted immunoviroplexes are an efficient siRNA delivery system for cancer therapy. Moreover, this study also suggests that a combination of conventional chemotherapy and tumor-directed anticancer siRNA therapy would be a better modality for cancer treatment.

In vitro and in vivo gene-transferring characteristics of novel cationic lipids, DMKD (O,O'-dimyristyl-N-lysyl aspartate) and DMKE (O,O'-dimyristyl-N-lysyl glutamate).

We chemically synthesized two different cationic lipids consisting of a core of lysine, two C-14 hydrocarbon chains, and either aspartatic acid or glutamic acid. The lipids were assigned the acronyms, DMKD and DMKE. Cationic liposomes prepared with the two different lipids were tested for their gene-transferring capabilities in various cell lines compared with that of control DOTAP liposomes. Under the same experimental conditions, the order of in vitro gene transfection efficiency was DMKE>or=DMKD>DOTAP. To identify the parameters influencing transfection efficiency, the DNA-binding affinities of the liposomes were compared and changes in particle size and surface charge were examined after complex formation. Both the DNA-binding affinity of the liposomes and the cell surface-binding affinity of the liposome-pDNA complexes were crucial for gene transfection. In addition, intravenously administered DMKE and DMKD liposomes exhibited different biodistribution characteristics and intensity of in vivo organ transfection from the DOTAP liposomes. Compared to the DOTAP liposomes, they were more readily transferred to the liver. Interestingly, when they were directly injected into tumor tissues, the DMKE lipoplexes were able to induce more efficient transgene expression in these tissues than the DOTAP and DMKD lipoplexes. This study suggests that a small difference in the cationic lipid backbone structure significantly affects gene-transferring capabilities. DMKE and DMKD liposomes can be utilized as efficient gene-transferring vehicles for hepatic or intra-tumoral gene transfection.

Inhibitory effect of the salmosin gene transferred by cationic liposomes on the progression of B16BL6 tumors.

Salmosin is a novel disintegrin containing the Arg-Gly-Asp sequence that significantly inhibits platelet aggregation, basic fibroblast growth factor-induced endothelial cell proliferation, and tumor progression by antagonizing integrin-mediated cell interactions. Previously, it was shown that daily administration of salmosin was able to inhibit tumor-derived angiogenesis and adherence and proliferation of tumor cells, resulting in suppression of tumor progression. However, it is very difficult to maintain a therapeutic level of salmosin in the blood by systemic administration of the protein. Hence, an alternative strategy for antiangiogenic cancer therapy, based on the in vivo expression of the salmosin gene administered with cationic liposomes, was investigated. The salmosin peptides expressed in vitro inhibited the proliferation of bovine capillary endothelial cells in a dose-dependent manner, presumably as a result of inhibition of cell adhesion mediated via alpha(v)beta(3) integrin. Subcutaneous administration of the salmosin gene resulted in systemic expression of the gene product and concomitant inhibition of the growth of B16BL6 melanoma cells. Suppression of pulmonary metastases, verified by experimental and spontaneous metastasis models in mice, also resulted from salmosin gene treatment. These results suggest that administration of the salmosin gene complexed to cationic liposomes is effective in maintaining antiangiogenic salmosin at an effective therapeutic level and may be clinically applicable to anticancer gene therapy.

Chitin-fibroin-hydroxyapatite membrane for guided bone regeneration: micro-computed tomography evaluation in a rat model.

BACKGROUND: In guided bone regeneration (GBR) technique, many materials have been used for improving biological effectiveness by adding on membranes. The new membrane which was constructed with chitin-fibroin-hydroxyapatite (CNF/HAP) was compared with a collagen membrane (Bio-Gide®) by means of micro-computed tomography. METHODS: Fifty-four rats were used in this study. A critical-sized (8 mm) bony defect was created in the calvaria with a trephine bur. The CNF/HAP membrane was prepared by thermally induced phase separation. In the experimental group (n = 18), the CNF/HAP membrane was used to cover the bony defect, and in the control group (n = 18), a resorbable collagen membrane (Bio-Gide®) was used. In the negative control group (n = 18), no membrane was used. In each group, six animals were euthanized at 2, 4, and 8 weeks after surgery. The specimens were analyzed using micro-CT. RESULTS: Bone volume (BV) and bone mineral density (BMD) of the new bone showed significant difference between the negative control group and membrane groups (P < 0.05). However, between two membranes, the difference was not significant. CONCLUSIONS: The CNF/HAP membrane has significant effect on the new bone formation and has the potential to be applied for guided bone regeneration.

Anti-EGFR immunonanoparticles containing IL12 and salmosin genes for targeted cancer gene therapy.

Tumor-directed gene delivery is of major interest in the field of cancer gene therapy. Varied functionalizations of non-viral vectors have been suggested to enhance tumor targetability. In the present study, we prepared two different types of anti-EGF receptor (EGFR) immunonanoparticles containing pDNA, neutrally charged liposomes and cationic lipoplexes, for tumor-directed transfection of cancer therapeutic genes. Even though both anti-EGFR immunonanoparticles had a high binding affinity to the EGFR-positive cancer cells, the anti-EGFR immunolipoplex formulation exhibited approximately 100-fold higher transfection to the target cells than anti-EGFR immunoliposomes. The lipoplex formulation also showed a higher transfection to SK-OV-3 tumor xenografts in mice. Thus, IL12 and/or salmosin genes were loaded in the anti-EGFR immunolipoplexes and intravenously administered to mice carrying SK-OV-3 tumors. Co-transfection of IL12 and salmosin genes using anti-EGFR immunolipoplexes significantly reduced tumor growth and pulmonary metastasis. Furthermore, combinatorial treatment with doxorubicin synergistically inhibited tumor growth. These results suggest that anti-EGFR immunolipoplexes containing pDNA encoding therapeutic genes could be utilized as a gene-transfer modality for cancer gene therapy.

Effect of vitamin D3 on production of progesterone in porcine granulosa cells by regulation of steroidogenic enzymes.

1,25-dihydroxyvitamin D3 (VD3), an active form of Vitamin D, is photosynthesized in the skin of vertebrates in response to solar ultraviolet B radiation (UV-B). VD3 deficiency can cause health problems such as immune disease, metabolic disease, and bone disorders. It has also been demonstrated that VD3 is involved in reproductive functions. Female sex hormones such as estrogen and progesterone are biosynthesized mainly in ovarian granulosa cells as the ovarian follicle develops. The functions of sex hormones include regulation of the estrus cycle and puberty as well as maintenance of pregnancy in females. In this study, we isolated granulosa cells from porcine ovaries and cultured them for experiments. To examine the effects of VD3 on ovarian granulosa cells, the mRNA and protein levels of genes were analyzed by Real-time PCR and Western blotting assay. Production of progesterone from granulosa cells was also measured by ELISA assay. As a result, transcriptional and translational regulation of progesterone biosynthesis-related genes in granulosa cells was significantly altered by VD3. Furthermore, progesterone concentrations in porcine granulosa cell-cultured media decreased in response to VD3. These results show that VD3 was a strong regulator of sex steroid hormone production in porcine granulosa cells, suggesting that vitamin D deficiency may result in inappropriate sexual development of industrial animals and eventually economic loss.

Insulin enhances RANKL-induced osteoclastogenesis via ERK1/2 activation and induction of NFATc1 and Atp6v0d2.

Insulin is one of the main factors affecting bone and energy metabolism, however, the direct effect of insulin on osteoclast differentiation remains unclear. Thus, in order to help elucidate that puzzle, the authors investigated the roles and regulatory mechanisms of insulin on osteoclasts differentiation. Co-stimulation with insulin and RANKL significantly enhanced the number of larger (>100 µm) osteoclastic cells and of TRAP-positive multinucleated cells compared with treatment by RANKL alone. Conversely, the insulin receptor shRNA markedly decreased osteoclast differentiation induced by insulin and RANKL. Insulin treatment significantly activated ERK1/2 MAP kinase as well as markedly induced the expression of NFATc1, an osteoclast marker gene, and Atp6v0d2, an osteoclast fusion-related gene. The pretreatment of PD98059, an ERK1/2 inhibitor, or insulin receptor shRNA effectively suppressed osteoclast differentiation and, in addition, blocked the expression of NFATc1 and Atp6vod2 induced by insulin stimulation. These data reveal insights into the regulation of osteoclast differentiation and fusion through ERK1/2 activation and the induction of NFATc1 and Atp6v0d2 by insulin.

Hepatotoxicity and nephrotoxicity of gallotannin-enriched extract isolated from Galla Rhois in ICR mice.

To evaluate the hepatotoxicity and nephrotoxicity of Galla Rhois (GR) toward the liver and kidney of ICR mice, alterations in related markers including body weight, organ weight, urine composition, liver pathology and kidney pathology were analyzed after oral administration of 250, 500 and 1,000 mg/kg body weight/day of gallotannin-enriched extract of GR (GEGR) for 14 days. GEGR contained 68.7±2.5% of gallotannin, 25.3±0.9% of gallic acid and 4.4±0.1% of methyl gallate. Also, the level of malondialdehyde (MDA), a marker of lipid peroxidation, was decreased with 19% in the serum of high dose GEGR (HGEGR)-treated mice. The body and organ weight, clinical phenotypes, urine parameters and mice mortality did not differ among GEGR-treated groups and the vehicle-treated group. Furthermore, no significant increase was observed in alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), blood urea nitrogen (BUN) and the serum creatinine (Cr) in the GEGR-treated group relative to the vehicle-treated group. Moreover, the specific pathological features induced by most toxic compounds such as CCl4 were not observed upon liver and kidney histological analysis. Overall, the results of the present study suggest that GEGR does not induce any specific toxicity in liver and kidney organs of ICR at doses of 1,000 mg/kg body weight/day, indicating that this is no observed adverse effect level (NOAEL).

Gene transfection by quantitatively reconstituted Sendai envelope proteins into liposomes.

Fusogenic liposomes (virosomes) consisting of Sendai virus envelope proteins have been utilized for in vitro and in vivo genetic modification of animal cells. In this study, the virosomes containing DNA were prepared by quantitative reconstitution of Sendai envelope proteins, fusion protein and hemagglutinin-neuramindase in liposomal vesicles. The Sendai virosomes more efficiently transferred genes into cultured 293 transformed kidney cells than 1,2-dioleoyl-3-(trimethylammonium) propane-based cationic liposomes. At 200:1 weight ratio of envelope protein and lipid, the virosomes exhibited the best efficiency of gene transfection into the cells. The Sendai virosomes required relatively a short period of incubation time and much less cytotoxic, compared to the cationic liposome/DNA complex. The transfection efficiency of the Sendai virosomes containing DNA was maintained 70% after a month. This type of Sendai virosomes is relatively convenient for preparation and storage, compared to fusogenic liposomes prepared by liposome-virus fusion. First of all, because the constituents are quantitatively formulated, this type of virosome formulation can provide further consistent transfection for gene therapy.

Inhibition of B16BL6 tumor progression by coadministration of recombinant angiostatin K1-3 and endostatin genes with cationic liposomes.

Transfection of the antiangiogenic angiostatin and endostatin genes was shown to be an alternative to high-dose administration of angiostatin or endostatin proteins for cancer therapy. We have systematically investigated whether coadministration of the mouse angiostatin kringle 1-3 gene (pFLAG-AngioK1/3) and the endostatin gene (pFLAG-Endo) complexed with cationic liposomes exhibits enhanced therapeutic efficacy. In vitro, the coexpressed mixture of angiostatin K1-3 and endostatin more effectively reduced angiogenesis in chorioallantoic membranes than either angiostatin K1-3 or endostatin alone. In vivo, subcutaneous co-administration of pFLAG-AngioK1/3 and pFLAG-Endo lipoplexes more effectively inhibited vascularization in Matrigel plugs implanted in mice than either one alone. Additionally, subcutaneous administration of these genes inhibited the growth and formation of pulmonary metastases of B16BL6 melanoma cells in mice. Compared to treatment with an empty vector, treatment with pFLAG-AngioK1/3 plus pFLAG-Endo inhibited 81% of tumor growth, while treatment with pFLAG-AngioK1/3 or pFLAG-Endo inhibited tumor growth 70 and 69%, respectively. Cotreatment with the two plasmids after primary tumor excision induced a 90% inhibition of pulmonary metastases versus 79% for pFLAG-AngioK1/3 or 80% for pFLAG-Endo individually. These results suggest that combined administration of angiostatin K1-3 and endostatin genes complexed with cationic liposomes may be an innovated antiangiogenic strategy for cancer therapy.

Inhibition of angiogenesis by salmosin expressed in vitro.

Recently, salmosin, a novel snake venom-derived disintegrin containing the Arg-Gly-Asp (RGD) sequence, was reported to be both antiangiogenic and antitumorigenic. The antitumor activity was substantiated by in vivo administration of recombinant salmosin into mice bearing tumors. However, it was difficult to prepare functionally active recombinant salmosin and to maintain a therapeutically effective concentration of the protein in the circulatory system by daily injections. Hence, we have suggested that salmosin gene transfer mediated by cationic liposomes may be a practical alternative for cancer treatment. Plasmids encoding the salmosin gene were constructed and then transferred by means of cationic liposomes into transformed human embryonic kidney (HEK) 293 cells. The transfected genes were able to produce functionally active salmosin proteins in vitro. In fact, the expressed salmosin remarkably inhibited proliferation of bovine capillary endothelial (BCE) cells and effectively inhibited the migration of highly metastatic B16BL6 mouse melanoma cells. Neovascularization in chick chorio-allantoic membranes (CAM) and in Matrigel implanted subcutaneously into mice was greatly inhibited in the presence of the expressed salmosin. Based on these experimental results, we suggest that the antitumor effect induced by salmosin gene transfection may be due to the antiangiogenic activity of the expressed salmosin proteins.

Effect of lipid compositions on gene transfer into 293 cells using Sendai F/HN-virosomes.

Fusogenic liposomes that incorporate Sendai virus envelope proteins, so-called Sendai virosomes, have been developed for in vitro and in vivo genetic modification of animal cells. In this study, several different virosomes of varying lipid compositions were formulated and their in vitro gene-transfer efficiencies compared. The virosomes were prepared by quantitative reconstitution of the Sendai envelope, fusion (F) and hemagglutinin-neuraminidase (HN) proteins into liposomal vesicles. Virosomes that contained luciferase reporter genes were tested in 293 transformed human kidney cells. F/HN-virosomes that were prepared with an artificial Sendai viral envelope (ASVE-virosomes) or phosphatidylserine (PS-virosomes) exhibited an 8- or 6-fold higher gene-transfer efficiency than cationic liposomes that were made with 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). F/HNvirosomes that were prepared with phosphatidic acid (PA-virosomes) instead of PS were less efficient in gene transfer than either ASVE- or PS-virosomes. In addition, the gene-transfer capability of ASVE- and PS-virosomes was maximal at a Ca2+ concentration of 510 mM. These results suggest that the incorporated lipid components significantly affect the in vitro gene transfer that is mediated by Sendai F/HN-virosomes.

Gene expression profiling in osteoclast precursors by insulin using microarray analysis.

The balance between bone formation by osteoblasts and destruction of mineralized bone matrix by osteoclasts is important for bone homeostasis. The increase of osteoclast differentiation by RANKL induces bone diseases such as osteoporosis. Recent studies have shown that insulin is one of main factors mediating the cross-talk between bone remodeling and energy metabolism. However, the systemic examination of insulin-induced differential gene expression profiles in osteoclasts has not been extensively studied. Here, we investigated the global effects of insulin on osteoclast precursors at the level of gene transcription by microarray analysis. The number of genes that were up-regulated by ≥ 1.5 fold after insulin treatment for 6 h, 12 h, or 24 h was 76, 73, and 39; and 96, 83, and 54 genes were down-regulated, respectively. The genes were classified by 20 biological processes or 24 molecular functions and the number of genes involved in 'development processes' and 'cell proliferation and differentiation' was 25 and 18, respectively, including Inhba, Socs, Plk3, Tnfsf4, and Plk1. The microarray results of these genes were verified by real-time RT-PCR analysis. We also compared the effects of insulin and RANKL on the expression of these genes. Most genes had a very similar pattern of expressions in insulin- and RANKL-treated cells. Interestingly, Tnfsf4 and Inhba genes were affected by insulin but not by RANKL. Taken together, these results suggest a potential role for insulin in osteoclast biology, thus contributing to the understanding of the pathogenesis and development of therapeutics for numerous bone and metabolic diseases.

Comparative study of chitosan/fibroin-hydroxyapatite and collagen membranes for guided bone regeneration in rat calvarial defects: micro-computed tomography analysis.

This study aimed to utilize micro-computed tomography (micro-CT) analysis to compare new bone formation in rat calvarial defects using chitosan/fibroin-hydroxyapatite (CFB-HAP) or collagen (Bio-Gide) membranes. Fifty-four (54) rats were studied. A circular bony defect (8 mm diameter) was formed in the centre of the calvaria using a trephine bur. The CFB-HAP membrane was prepared by thermally induced phase separation. In the experimental group (n=18), the CFB-HAP membrane was used to cover the bony defect, and in the control group (n=18), a resorbable collagen membrane (Bio-Gide) was used. In the negative control group (n=18), no membrane was used. In each group, six animals were euthanized at 2, 4 and 8 weeks after surgery. The specimens were then analysed using micro-CT. There were significant differences in bone volume (BV) and bone mineral density (BMD) (P<0.05) between the negative control group and the membrane groups. However, there were no significant differences between the CFB-HAP group and the collagen group. We concluded that the CFB-HAP membrane has significant potential as a guided bone regeneration (GBR) membrane.