Hiroyuki Oura

Fabrication of biodegradable scaffolds by use of self-assembled magnetic sugar particles as a casting template

Technologies to develop scaffolds with controlled pore layout and porosity have great significance in tissue engineering. As one method of scaffold fabrication, porogen leaching has been commonly used to control pore size, pore structure and porosity in the scaffold. In this paper, we describe a novel approach to fabricate 2D and 3D porous biodegradable scaffolds made of poly(L-lactide-co-epsiv-caprolactone) by using magnetic sugar particles as porogens. First, ferrite micro/nano particles were encapsulated in sugar microspheres to make them magnetized. After sieving magnetic sugar particles, those diameter-controlled particles were attracted by a magnetic force to form an assembled template for polymer casting. A magnetic field with polka-dot pattern was also utilized to align particles on desired positions. After polymer casting and removal of the sugar template, spherical pores were generated inside scaffold. For future application in vascular tissue engineering, we extended the scaffold fabrication to straight tubular scaffolds by winding 2D porous sheets on sacrificial molds. The biocompatibility of the developed scaffold was confirmed by viable cells after 4-day culture.

Fabrication of Cell-Adhesion Surface and Capillary Vessel Model by Photolithography

We have been developing scaffolds of three-dimensional (3D) synthetic vascular prosthesis in tailor-made. Human umbilical vein endothelial cells (HUVECs) attached on the inner surface of the scaffold have anticoagulant effects. Asperity structures of the inner surface are important to cell adhesion. It is important to quantify the inner surface asperity condition of the scaffold by observing HUVECs behavior and morphology. For this purpose, we recreated the inner surface profile of the scaffold on a poly(dimethilsiloxane) (PDMS) substrate by microfabrication. We made semiround convex patterns of resist that had 8 mum in diameter and 5 mum high using photolithography, and the concave pattern on the PDMS substrate by printing. We observed HUVECs adhering to the PDMS substrate having concave pattern on it surface. The distribution density of the concaves of the tested pattern is 1600 /mm2 or 40,000 in a 25 mm2 area. In addition, we fabricated a capillary vessel model by photolithography, creating a branched capillary tube model that had 13 mum in diameter. We confirmed that the capillary vessel model had no leakage using a methylene blue solution flow in the channel.

Development of Multi-Layer Scaffolds Based on Artificial Configuration

In this work, we propose a multi-layered scaffold made of elastic biodegradable polymer PLCL [1] by salt leaching method [2], which has high porosity at inner layer and high stiffness in outer layer. Before implanting to a damaged position of a patient, scaffolds should be coated with human umbilical vein endothelial cells (HUVECs). (Porous structure is also served as a pathway of sufficient nutrients and oxygen). This inner layer has porosity as means to culture HUVECs on itself, and outer layer has stiffness as means to maintain shape of scaffolds against blood flow as that of blood vessels does. These inner and outer layers were reproduced also as means of intimal layer and internal elastic lamina in human blood vessel especially muscular artery respectively. It was also verified that this fabrication method can be applied to fabricate tri-layered tubular artificial blood vessel scaffold by SEM. Mechanical characterization and porosity of bi-layered scaffolds were evaluated by tensile test and image processing respectively. These parameters were confirmed that can be controllable by adjusting concentration of NaCl for inner layer used in salt leaching method. HUVECs were cultured on the porous structure of bi-layered scaffolds, and the scaffolds biocompatibility was confirmed by viability of cells. Results obtained from these experiments shows we can propose artificial blood vessel scaffold, which has optimal porosity and mechanical strength, not to induce blood clot, intimal hypertrophy and so on in patients body.

Development of biodegradable scaffolds by leaching self-assembled magnetic sugar particles

Technologies to develop scaffolds with controlled diameter and high porosity have great significance in tissue engineering. We have fabricated biodegradable 2D and 3D scaffolds with ordered array of pores by casting polymer on self-assembled d-fructose (sugar) microspheres. First, ferrite microparticles were encapsulated in sugar spheres to make them become magnetized. After sieving magnetic sugar particles, those diameter-controlled particles were attracted by a magnet to form a self-assembled template for polymer casting. Interspaces of self-assembled template were successfully infiltrated with polymer. After removal of sugar particles, ordered array of pores were generated on the surface of scaffolds. Elastic modulus of a sheet-like scaffold was measured to be 2.0 MPa and was reasonably lower than that of a nonporous sheet. The biocompatibility of the developed scaffold was confirmed by the viability of human umbilical vein endothelial cells.

Multi-scale transparent arteriole and capillary vessel models for circulation type blood vessel simulator

We proposed a fabrication method for multiscale transparent arteriole and capillary vessel models and demonstrated the fabrication of microchannels with circular cross sections φ10 – 500 µm. First, we demonstrated fabrication of φ10 – 500 µm arteriole and capillary vessel block models using photolithography. The circularity of fabricated φ10, 50, and 500 µm microchannels were 84.0%, 61.5%, and 82.3%, respectively. Flow experiments demonstrated that these channels had no leakage. Next, we proposed a fabrication method for φ100 – 500 µm arteriole membrane models, which connect larger membrane models and smaller block models. These models were prepared with grayscale lithography and a wax and PVA (polyvinyl alcohol) mixture material. The proposed approach overcame the brittleness of a previous sacrificial model fabricated by ink jet rapid prototyping. The membrane model had a circular cross section with a channel circularity of 90%. Finally, we succeeded in making transparent membranous and block arteriole model with which we can simulate blood circulation.

Feedback Control with Hybrid Pump for Realistic Human Blood Pressure Reconstruction

Abstract Human blood pressure simulation is of particular interest for artificial blood vessel evaluation in regenerative medicine, and for endovascular surgery simulation to become more realistic. Conventional pumps do not offer a solution for the flow quality requirements; therefore we propose a hybrid pump that combines the best properties of lobe and piston pumps. Feedback control was applied to the hybrid pump, the system was tested using three different waveforms as reference. One of the waveforms is a polynomial approximation of pressure variation inside a coronary artery; when used a realistic human blood pressure simulation was produced. A scaffold for blood vessel regeneration was evaluated with the simulated human blood pressure.

Fabrication of Transparent Arteriole Membrane Models

We made transparent arteriole membranous models by using grayscale lithography. We employed a sacrificial model made of WAX and PVA mixture as a novel molding material. Our goal is to complement previous surgical simulators for practice and rehearsal of medical treatments. Since block vessel models cannot recreate moderate compliance which is similar to that of the real blood vessel, here we propose fabrication method for transparent arteriole membranous model which has circular cross section smaller than 500 mum in diameter. Fabrication method of the model as well as evaluation result of the molding material is reported.

Construction of Biodegradable Polymer Scaffold by Photolithography

In this study, we succeeded in fine construction of the PLCL (poly-L-lactide-cocaprolactone) scaffold with micrometer range resolution using photolithography. PLCL is a biodegradable polymer which is used as a material of the scaffold to reproduce the blood vessel. Photolithography known as a technique of microfabrication was used for the processing of PLCL. By this technique, we fabricated a sheet-like PLCL scaffold which is patterned straight line ditches. Laminating the sheet-like scaffold, we fabricated microchannels in the PLCL scaffold. By the attachment of HUVECs in the channel, biocompatibility of the sheet-like PLCL scaffold was confirmed. It is expected to evaluate the behavior of cells in the scaffold.

Patient-Specific Blood Vessel Scaffold for Regenerative Medicine

In this research, we propose a method to construct an artificial blood vessel scaffold with patient-specific complex 3-dimensional shape and biocompatible porous polymer membrane adapted for cell cultivation, by introducing salt leaching technique into a fabrication technique for patient-specific 3D blood vessel model proposed by authors. In this method, a membranous PVA (polyvinyl alcohol) structure of desired 3-dimensional blood vessel shape was fabricated based on CT data. And the PVA structure was dip coated with polymer solution made by dissolving the Caprolactone (3wt%) and NaCl particle (27wt%) into chloroform (organic solvent), Finally, the NaCl particle and the membranous PVA structure were both eluted from the structure by dissolving these materials under water, and finally a patient-specific blood vessel scaffold with desired 3D vascular shape was constructed. Presented scaffold has the mechanical compliance similar to human blood vessel and provides porous polymer structure appropriate for cell cultivation.