[Ectopic osteogenesis of immortalized human bone marrow mesenchymal stem cells and heterogeneous bone]

To provide the seed cells for bone tissue engineering, to establish immortalized human bone marrow mesenchymal stem cells (MSCxj) and to investigate the ectopic osteogenesis of MSCxj.

MSCxjs of the 35th and 128th generations were maintained and harvested when the cell density reached 2 x 10(9). Then, these cells were co-cultured with heterogeneous bone scaffold in groups A (the 35th generation, n = 12) and group B (the 128th generation, n = 12); heterogeneous bone alone was used in group C (n = 12). The cell proliferation was observed by scanning electron microscopy (SEM) after 48 hours and 18 days of osteogenic induction culture. The complex was implanted subcutaneously through a 3-mm-incision at both sides of the back in 18 nude mice. Tetracycline labeling was performed before the animals were sacrificed. Tetracycline fluorescence staining, HE staining, ponceau staining, and immunohistochemistry staining for osteocalcin were performed at 4, 8, and 12 weeks after transplantation; the morphologic quantitative analysis was made.

After 48 hours, SEM showed that MSCxjs adhered to heterogeneous bone and grew well; after 18 days, a large number of new filamentous extracellular matrix and small granules were found to cover the cells. The results of tetracycline fluorescence staining, HE staining, and ponceau staining in groups A and B showed that the osteogenesis was not obvious at 4 weeks after transplantation; osteoid matrix deposition was noted around and in the heterogeneous bone at 8 weeks; and osteogenesis was increased at 12 weeks. There was no significant difference in bone formation between groups A and B. Osteogenesis was not observed in group C. The osteocalcin expressions were positive in groups A and B. The bone ingrown percentages of groups A and B were 5.64% +/- 2.68% and 4.92% +/- 2.95% at 8 weeks, and 13.94% +/- 2.21% and 14.34% +/- 3.46% at 12 weeks, showing significant differences between 8 weeks and 12 weeks at the same group (P < 0.05) and no significant difference between groups A and B at the same time (P > 0.05).

MSCxj has favorable abilities of ectopic osteogenesis and can be applied as seeded cells in bone tissue engineering.