Human mesenchymal stem cells (hMSCs) are multipotent cells, and have been expanded and differentiated into several kinds of mesodermal tissue in vitro. In order to promote bone repair, enhancement of the proliferation and differentiation of hMSCs into osteoblasts in vitro is recommended prior to therapeutic delivery. However, for clinical applications, it is still unclear which method is more advanced for tissue engineering: to transplant undifferentiated cells or partially differentiated stem cells. Therefore, the present study aimed to investigate how osteogenic differentiation medium (ODM) affects hMSCs cultured in a 3D scaffold using a radial-flow bioreactor (RFB) besides cell growth medium (GM). To produce precultured sheets, the hMSCs were first seeded onto type 1 collagen sheets and incubated for 12 h, after which they were placed in the RFB for scaffold fabrication. The culture medium was circulated at 3 mL/min and the cells dynamically cultured for 1 week at 37 °C. Static cultivation in a culture dish was also carried out. Cell proliferations were evaluated by histological analysis and DNA-based cell count. Alkaline phosphatase (ALP) activity, immunocytochemical analysis with BMP-2, and osteopontin on the hMSCs in the collagen scaffold were performed. After 14 days of ODM culture, a significant increase in cell number and a higher density of cell distribution in the scaffold were observed after both static and dynamic cultivation compared to GM culture. A significant increase in ALP activity after 14 days of ODM was recognized in dynamic cultivation compared with that of static cultivation. Cells that BMP-2 expressed were frequently observed after 14 days in dynamic culture compared with other conditions, and the expression of osteopontin was confirmed in dynamic cultivation after both 7 days and 14 days. The results of this study revealed that both the proliferation and bone differentiation of hMSCs in 3D culture by RFB were accelerated by culture in osteogenic differentiation medium, suggesting an advantageous future clinical applications for RFB cell culture and cell transplantation for tissue engineering.

Dynamic cultivation using a radial flow bioreactor (RFB) has gained increasing interest as a method of achieving bone regeneration. In order to enhance bone generation in large bone defects, it is necessary to use an RFB to expand the primary cells such as bone marrow cells derived from biotissue. The present study aimed to evaluate the cell expansion and osteogenic differentiation of rat bone marrow cells (rBMC) when added to basic fibroblast growth factor containing medium (bFGFM) or osteogenic differentiation factor containing medium (ODM) under dynamic cultivation using an RFB. Cell proliferation was evaluated with a DNA-based cell count method and histological analysis. An alkaline phosphatase (ALP) activity assay and immunohistochemistry staining of osteogenic markers including BMP-2 and osteopontin were used to assess osteogenic differentiation ability. After culture for one week, rBMC cell numbers increased significantly under dynamic cultivation compared with that under static cultivation in all culture media. For different culture media in dynamic cultivation, bFGFM had the highest increase in cell numbers. ALP activity was facilitated by dynamic cultivation with ODM. Furthermore, both BMP-2 and osteopontin were detected in the dynamic cultivation with ODM. These results suggested that bFGFM promotes cell proliferation and ODM promotes osteogenic differentiation of rBMC under dynamic cultivation using an RFB.