Masafumi Oda

Liver-targeted hydrodynamic gene therapy: Recent advances in the technique

One of the major research focuses in the field of gene therapy is the development of clinically applicable, safe, and effective gene-delivery methods. Since the first case of human gene therapy was performed in 1990, a number of gene-delivery methods have been developed, evaluated for efficacy and safety, and modified for human application. To date, viral-vector-mediated deliveries have shown effective therapeutic results. However, the risk of lethal immune response and carcinogenesis have been reported, and it is still controversial to be applied as a standard therapeutic option. On the other hand, delivery methods for nonviral vector systems have been developed, extensively studied, and utilized in in vivo gene-transfer studies. Compared to viral-vector mediated gene transfer, nonviral systems have less risk of biological reactions. However, the lower gene-transfer efficiency was a critical hurdle for applying them to human gene therapy. Among a number of nonviral vector systems, our studies focus on hydrodynamic gene delivery to utilize physical force to deliver naked DNA into the cells in the living animals. This method achieves a high gene-transfer level by DNA solution injections into the tail vein of rodents, especially in the liver. With the development of genome editing methods, in vivo gene-transfer therapy using this method is currently the focus in this research field. This review explains the method principle, efficiency, safety, and procedural modifications to achieve a high level of reproducibility in large-animal models.

An atmospheric‐pressure plasma‐treated titanium surface potentially supports initial cell adhesion, growth, and differentiation of cultured human prenatal‐derived osteoblastic cells

An atmospheric-pressure plasma (APP) treatment was recently reported to render titanium (Ti) surfaces more suitable for osteoblastic cell proliferation and osteogenesis. However, the mechanism of action remains to be clearly demonstrated. In this study, we focused on cell adhesion and examined the effects of the APP treatment on the initial responses of human prenatal-derived osteoblastic cells incubated on chemically polished commercially pure Ti (CP-cpTi) plates. In the medium containing 1% fetal bovine serum, the initial cell adhesion and the actin polymerization were evaluated by scanning electron microscopy and fluorescence microscopy. The expression of cell adhesion-related molecules and osteoblast markers at the messenger RNA level was assessed by real-time quantitative polymerase chain reaction. Although the cells on the APP-treated CP-cpTi surface developed fewer cytoskeletal actin fibers, they attached with higher affinity and consequently proliferated more actively (1.46-fold over control at 72 h). However, most of the cell adhesion molecule genes were significantly downregulated (from 40 to 85% of control) in the cells incubated on the APP-treated CP-cpTi surface at 24 h. Similarly, the osteoblast marker genes were significantly downregulated (from 49 to 63% of control) at 72 h. However, the osteoblast marker genes were drastically upregulated (from 197 to 296% of control) in these cells by dexamethasone and β-glycerophosphate treatment. These findings suggest that the APP treatment improves the ability of the CP-cpTi surface to support osteoblastic proliferation by enhancing the initial cell adhesion and supports osteoblastic differentiation when immature osteoblasts begin the differentiation process.

Quantitative single-cell motility analysis of platelet-rich plasma-treated endothelial cells in vitro.

Platelet‐rich plasma (PRP) has been widely applied in regenerative therapy due to its high concentration of growth factors. Previous in vitro and in vivo studies have provided evidence supporting the angiogenic activity of PRP. To more directly demonstrate how PRP acts on endothelial cells, we examined the PRP‐induced changes in the motility of human umbilical vein endothelial cells by examining the involvement of VEGF. Time‐lapse quantitative imaging demonstrated that in the initial phase (∼2 h) of treatment, PRP substantially stimulated cell migration in a wound‐healing assay. However, this effect of PRP was not sustained at significant levels beyond the initial phase. The average net distance of cell migration at 10 h was 0.45 ± 0.16 mm and 0.82 ± 0.23 mm in control and PRP‐stimulated cells, respectively. This effect was also demonstrated with recombinant human VEGF and was significantly attenuated by a neutralizing anti‐VEGF antibody. Immunofluorescent examination of paxillin and actin fibers demonstrated that PRP concomitantly up‐regulated focal adhesion and cytoskeletal formation. Western blotting analysis of phosphorylated VEGFR2 demonstrated that PRP mainly stimulated the phosphorylation of immature VEGFR2 in a dose‐ and time‐dependent manner, an action that was completely blocked by the neutralizing antibody. Taken together, these data suggest that PRP acts directly on endothelial cells via the activation of VEGFR2 to transiently up‐regulate their motility. Thus, the possibility that PRP desensitizes target endothelial cells for a relatively long period of time after short‐term activation should be considered when the controlled release system of PRP components is designed.

267. Image-Guided, Liver-Targeted Hydrodynamic Gene Delivery in Dogs – Preclinical Assessment of Effectiveness and Safety of the Procedure

Image-guided, liver-targeted hydrodynamic gene delivery was evaluated in dogs to assess the safety and effectiveness of the procedure for human clinical trial. The procedure involves image-guided catheter insertion to hepatic veins in the dogs followed by computer-controlled hydrodynamic gene delivery using newly developed injector for clinical use. Optimum parameters for the procedure, including the location of the balloon catheter in the hepatic vein, intravascular pressure upon injection, injection volume, and sequential injections to multiple lobes of the injected liver, were employed as previously we reported. Hydrodynamic injection of human factor IX expressing plasmid (pBS-hFIX) was performed in 7 dogs (female, 20 kg) and short- and long-term effects of the procedure were assessed. We demonstrate that plasma level of human factor IX and coagulation activity in dogs increased from the background level at ~20 ng/ml and ~8% to 21768.1 ng/ml and 36.8%, respectively in 1 week after hydrodynamic gene delivery and sustained for 4 weeks. Transgene expression in the hepatocytes was also confirmed by immunohistochemical stainings. For safety assessment, we demonstrated that the impacts of image-guided liver-targeted hydrodynamic gene delivery are localized in the site of injection in the liver. Histological analysis showed significant expansion of sinusoids at the injected site to 190% of their original size. Hepatic microcirculation analysis of liver-targeted hydrodynamic injection using reflectance spectrophotometry showed significant decrease of the oxygen saturation of the Hb in the blood of the injected lobe from 30 to 0 unit upon the injection and recovered smoothly after the injection. Serum analysis showed a transient, 10- to 20-fold increase in hepatobiliary enzymes, including aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase after hydrodynamic injections, which recovered within 4 days. We also explored for the first time, the levels of the cytokines following the hydrodynamic injections to the large animals. There was no increase in the systemic inflammatory cytokines of IFN-γ, IL-8, IL-18, and IL-4, although an increase in serum levels of TNF-α, IL-10, MCP-1, canine KC, and IL-6, which were related to vascular stretching representing the sinusoidal expansion was observed. No impacts on their respiratory, cardiovascular conditions, or long-term body weight changes were observed throughout the study. These results of preclinical assessments support the clinical applications of image-guided, liver-targeted hydrodynamic gene delivery.This work was supported in part by NIH grants RO1EB002946 and RO1HL075542, and by Japanese Society for the Promotion of Science Grants 22890064, 23790595, and 26860354.

Measuring the 3D-Position of Cementless Hip Implants using Pre- and Postoperative CT Images

Quantitative measurement of the location of cementless hip implants postoperatively not only explains subsequent common complications, such as impingement and loosening, but can also predict postoperative subject-specific primary stability, if combined with the finite element method. However, the metal artifacts on the postoperative computed tomography (CT) image make it difficult to estimate the threedimensional (3D) alignment, implant shape, bone geometry, and mineral density precisely. Therefore, we developed a novel, accurate measuring technique for the 3D implant position using pre- and postoperative CT images, which is based on a 3D surface registration algorithm. We can obtain a computer model of the femur from preoperative CT data combined with a stem model from computer-assisted design (CAD) data. This study assessed the accuracy of our novel technique for the 3D estimation of implant position. Five sawbones femurs and five cementless stems were used. Ceramic sphere markers were bonded to the bone and stem surface to define local coordinate systems for the femur and stem, respectively. Each femur underwent CT at slice pitches of 1, 2, and 3 mm before and after stem implantation. The relative positions of the stem and femur obtained using our method were compared with those measured using a 3D laser digitizer, used as the true value. The relative estimated position errors were within 0.3 mm in translation and 0.4° in rotation, regardless of slice pitch. From the viewpoint of pixel size of the CT image in a clinical situation, the estimation is sufficiently accurate. Therefore, our measurement technique is useful for quantifying the relative 3D position of implants to the bone geometry and for performing comparative studies among subjects.

Tribological Evaluation of Boriding Metal by Acoustic Emission Technique.

An acoustic emission technique was developed to study the tribological characteristic of boriding SKD steel using a pin-on-disk type wear testing machine with acoustic emission sensor. AE signal of 300 kHz frequency spectrum generated by crack propagation in the specimens was observed in the wear tesing. The R. M. S. value of the emission signal was expressed in terms of wear and friction parameters, The experimental results showed that AE generation from pin contact with a rotating disk indicates the feasibility of utilizing AE in boriding metal wear sensing.