Research on Fabrication of Tissue Engineering Scaffold for Reconstruction of Internal Organs with High Metabolic Rate by Laser Sintering Solid Freeform Fabrication and Cell Culture

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Reconstruction of internal organs such as liver requires three dimensional scaffolds that are equipped with network of fine flow channels. To develop such scaffolds, we applied laser sintering freeform fabrication method using biodegradable plastic powder. This method provides a large choice of material as its advantage, but on the other hand, precision of commercially available machines is not sufficient for our purpose. To avoid this drawback, we developed a new apparatus to reduce laser spot size on the powder bed by a factor of 70 to 50%. Additionally, we investigated relationship between precision of fabrication and various properties of water leachable filler, which is applied to provide the scaffolds with very high porosity around 90%. As a result of the parameter optimization and use of the new apparatus, we succeeded in doubling the precision. The newly developed scaffold was equipped with a network of flow channels of which thickness is 500um at the minimum. This improvement … More is expected to increase cell density to be obtained by a factor of around 4. Generally, reducing focal spot size of a laser beam sacrifices the scanning range of laser scanner by optical reason. To retrieve the lost build envelope, we develop a new system that can move the laser scanner in a plane parallel to the powder bed. Resultantly, wide envelope of 150×150mm^2 is obtained although employed galvanomirror type laser scanner's range is as narrow as 50×50mm^2 to realize fabrication of large scaled scaffolds. Using this equipment, we succeeded in developing a porous body with flow channels.To avoid the problem of insufficient oxygen delivery following scaling up of scaffold, feasibility of using nano-encapsulated hemoglobin (artificial red blood cells) was investigated. In culture test using hepatic progenitor cells, a certain improvement of oxygen supply was found. However, cytotoxicity caused by free hemoglobin molecules released from the capsules that were ingested and broken in the cells was observed, although encapsulation of hemoglobin successfully prevented this problem in preliminary test using mature hepatic cells. Perfusion culture of human liver cancer cell lines using a developed scaffold with flow channel network was tested, and significant improvement both in cell growth and expression of liver function was observed. In the test, cell propagation was limited in the range of 200μm from each flow channel. This demonstrates that optimal scaffold should be so designed that any position in the scaffold is in the range of 200μm from a flow channel. Less