Masashige Shinkai

Functional magnetic particles for medical application

Magnetic particles for medical applications have been developed by many researchers. Since magnetic particles have unique magnetic features not present in other materials, they can be applied to special medical techniques. Separation, immunoassay, magnetic resonance imaging (MRI), drug delivery, and hyperthermia are enhanced by the use of magnetic particles. Magnetite cationic liposomes (MCLs), one of the group of cationic magnetic particles, can be used as carriers to introduce DNA into cells since their positively charged surface associates with the negatively charged DNA. They can also be used as heat mediators for cancer therapy. Magnetic particles conjugated with tumor-specific antibodies have enabled tumor-specific contrast enhancement in MRI. In addition, antibody-conjugated magnetic particles were shown to target renal cell carcinoma cells, and are applicable to the hyperthermic treatment of carcinomas. The use of magnetic particles with their unique features will further improve medical techniques.

Icariin induces osteogenic differentiation in vitro in a BMP- and Runx2-dependent manner

To effectively treat bone diseases using bone regenerative medicine, there is an urgent need to develop safe cheap drugs that can potently induce bone formation. Here, we demonstrate the osteogenic effect of icariin, the main active compound of Epimedium pubescens. Icariin induced osteogenic differentiation in pre-osteoblastic MC3T3-E1 cells and mouse primary osteoblasts. Icariin upregulated the mRNA expression levels of the osteoblast marker genes runt-related transcription factor 2 (Runx2) and inhibitor of DNA-binding 1 (Id-1). The osteogenic effect was inhibited by the introduction of Smad6 or dominant-negative Runx2, as well as Noggin treatment. Furthermore, icariin induced the mRNA expression of bone morphogenetic protein (BMP)-4. These data suggest that icariin exerts its potent osteogenic effect through induction of Runx2 expression, production of BMP-4 and activation of BMP signaling. The extremely low cost of icariin and its high abundance make it appealing for bone regenerative medicine.

Bone regeneration using collagen type I vitrigel with bone morphogenetic protein-2.

Bone morphogenetic protein-2 is a very promising candidate for the treatment of bone diseases and defects, but more effective therapeutic methods are required due to its instability in vivo. A controlled and localized delivery system of Bone morphogenetic protein-2 would be appropriate for effective bone regeneration. Here, we report a novel delivery system of bone morphogenetic protein-2 using vitrigel (a novel stable collagen gel membrane prepared from vitrified type I collagen) for in vivo bone regeneration. Scanning electron microscopy revealed that the collagen vitrigel formed a tightly woven network with average pore sizes of about 1-2 microm. The vitrigel scaffold delivery system exhibited sustained release of bone morphogenetic protein-2 and >80% of the total bone morphogenetic protein-2 was still retained in the vitrigel after 15 days in phosphate-buffered saline in vitro. Bone morphogenetic protein-2-containing vitrigel was transplanted into mouse calvarial defects. The enhanced mechanical strength of the vitrigel made it easier to implant into defects without damage. Obvious bone regeneration was observed in the defects of mice treated with as little as 0.19 microg of bone morphogenetic protein-2 at 4 weeks after the transplantation. The local and sustained delivery system for bone morphogenetic protein-2 developed in the present study may represent a powerful modality for bone regeneration.

Recombinant extracellular matrix-like proteins with repetitive elastin or collagen-like functional motifs

Using overlap elongation PCR, we created repetitive DNA libraries encoding the elastin VPGVG and collagen-like GERGDRGDP sequences. From these libraries we isolated two repetitive DNA sequences, Col-5 encoding [(GERGDRGDP)5GER], and Ela-16 encoding [(VPGVG)16VPG]. Both proteins were expressed as thioredoxin fusion proteins. The resulting recombinant extracellular matrix-like proteins had the expected properties (cell adhesive ability and thermally responsive structural change) of the functional motif sequence unit used.

Cell transfer printing from patterned poly(ethylene glycol)-oleyl surfaces to biological hydrogels for rapid and efficient cell micropatterning.

Cell transfer printing from patterned poly(ethylene glycol)‐oleyl surfaces onto biological hydrogel sheets is investigated herein, as a new cell stamping method for both cell microarray and tissue engineering. By overlaying a hydrogel sheet on the cells immobilized on the poly(ethylene glycol)‐oleyl surface and successively peeling it off, the immobilized cells were transferred onto a hydrogel sheet because the adhesive interaction between the cells and the hydrogel was stronger than that between the cells and the poly(ethylene glycol)‐oleyl surface. Four types of human cell could be efficiently transferred onto a rigid collagen sheet. The transfer printing ratios, for all cells, were above 80% and achieved within 90u2009min. A cell microarray was successfully prepared on a collagen gel sheet using the present stamping method. We have also demonstrated that the transferred pattern of endothelial cells is transformed to the patterned tube‐like structure on the reconstituted basement membrane matrix. Finally, the patterns of two types of endothelial cell are shown to be easily prepared on the matrix, and the desired tube‐like structures, including the orderly pattern of the two different cells, were formed spontaneously. Thus, the present poly(ethylene glycol)‐oleyl coated substrates are useful for rapid and efficient cell stamping, in the preparation of multi‐cellular pattern on extracellular matrices. Biotechnol. Bioeng. 2012;109: 244–251.

Local hyperthermia enhances thrombosis in aneurysms containing platinum coils.

Despite recent technical advances in embolization of cerebral aneurysms with platinum coils, some aneurysms eventually resulted in incomplete packing with remnant neck or dome filling. Such a situation with a remaining inflow zone may pose a risk of rupture and subsequent regrowth. Metals characteristically generate heat under high-frequency alternating magnetic fields (AMF). We used this property to induce local hyperthermia and promote thrombogenesis in incompletely packed aneurysms. Glass model aneurysms packed with coils were subjected to AMF to investigate the correlation between weight of platinum and temperature elevation and the correlation between flow rates of water through the model and temperature elevation. Next, activated coagulation time (ACT) of blood obtained from dogs was studied at various temperatures. Finally, side-wall aneurysms created in the canine carotid artery using a venous patch were packed with platinum coils. Change in temperature and angiographic changes were investigated after AMF application. In the glass model, the weight of platinum was correlated with elevation of temperature, and a negative logarithmic correlation was evident between flow rate and elevation of temperature. Elevation of blood sample temperature tended to shorten ACT. In canine carotid aneurysms, elevation of intra-aneurysmal temperature was confirmed and sufficient elevation of temperature was found to promote angiographically evident thrombogenesis of the remnant space after AMF application. Local hyperthermia may be useful in completing luminal obliteration of aneurysms after coil embolization. It may particularly useful for ruptured aneurysms to prevent the early rerupture.