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

肝脏等内部器官的重建需要三维支架，这些支架配备了细小的流动通道网络。为了开发这种支架，我们采用了激光烧结自由成型的方法，使用可生物降解的塑料粉末。这种方法的优点是提供了大量的材料选择，但另一方面，商业上可用的机器的精度不足以满足我们的目的。为了避免这一缺点，我们开发了一种新的装置，将粉床上的激光光斑尺寸减小了70%到50%。此外，我们还研究了可渗水填料的各种性能与制备精度的关系，该填料可用于提供孔隙率高达90%左右的支架。由于参数的优化和新仪器的使用，我们成功地将精度提高了一倍。新开发的支架配备了最小厚度为500um的流动通道网络。这一改进…一般情况下，减小激光束的焦斑尺寸会牺牲激光扫描仪的扫描范围。为了找回丢失的建筑封套，我们开发了一种新的系统，可以在平行于粉末床的平面上移动激光扫描仪。结果表明，虽然采用电镜式激光扫描仪实现了大尺寸支架的制作，但仍获得了150×150 mm~2的宽包络。利用该设备成功地研制出具有流道的多孔体。为了避免支架放大后氧气输送不足的问题，研究了使用纳米包裹的血红蛋白(人工红细胞)的可行性。在肝祖细胞的培养试验中，发现供氧量有一定的改善。然而，观察到从胶囊中释放的游离血红蛋白分子在细胞内被摄取和破裂时所引起的细胞毒性，尽管在使用成熟肝细胞进行的初步试验中，包裹血红蛋白成功地避免了这一问题。用研制的流道网络支架对人肝癌细胞系进行灌流培养，观察到细胞生长和肝功能表达均有明显改善。在试验中，细胞繁殖被限制在每个流动通道200μm的范围内。这表明，最优脚手架的设计应该使脚手架中的任何位置都在距流道200μm的范围内。较少

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