A Novel Cell-Loading Method into Porous β-TCP Blocks

In the field of bone tissue engineering using cells combined with scaffolds, it is important to efficiently load cells into porous scaffolds. We devised a novel cell-loading method into porous beta-tricalcium phosphate (β-TCP) blocks. In this study, we compared this method with two conventional cell-loading methods in terms of cell-loading efficiency and in vivo bone formation capability. Bone marrow stromal cells (BMSCs) were obtained from the femurs of Fisher rats. After about 10 days of culture, BMSCs were harvested and suspended in the plasma of the Fisher rats at a concentration of 2×106 cells/ml. This cell suspension was loaded into porous β-TCP cubes (5×5×5mm) by using three loading methods: a soaking method, a post low-pressure method and a pre low-pressure method (the novel method). These β-TCP cubes were cross-sectioned and stained with toluidine blue and cell-counted. Cell-loading efficiency was significantly higher when using the novel methods. For the study of in vivo bone formation capability, nearly confluent BMSCs were exposed in an osteogenic medium supplemented with 10-7 M dexamethasone, 50µg/ml L-ascorbic acid phosphate and 10mM β-glycerophosphate for 4 days. These osteogenic cells were harvested and suspended in the plasma of the Fisher rats at a concentration of 2×106 cells/ml. This cell suspension was loaded into porous β-TCP cubes (5×5×5mm) by using the three cell-loading methods. Immediately, these β-TCP cubes were implanted at subcutaneous sites in the backs of 7-week-old male Fisher rats and harvested at postoperative 3 and 6 weeks. After cross-sectioning, these sections were stained with hematoxylin and eosin, and the new bone formation area was quantified. Consistent with cell-loading efficiency, in vivo bone formation capability was significantly higher in the novel method at postoperative 6 weeks. We showed the usefulness of the novel cell-loading method in bone tissue engineering.