Proliferation and osteogenesis of immortalized bone marrow-derived mesenchymal stem cells in porous polylactic glycolic acid scaffolds under perfusion culture

摘要

Human bone marrow mesenchymal stem cells (hMSCs) are promising candidates for cell therapy and tissue engineering. However, the life span of hMSCs during in vitro culture is limited. Human telomerase catalytic subunit (hTERT) gene transduction could prolong the life span of hMSCs and maintain their potential of osteogenic differentiation. Therefore, hMSCs transduced with hTERT (hTERT-hMSCs) could be used as a cell model for in vitro tissue engineering experiment because of its prolonged life span and normal cellular properties. A perfusion culture system for proliferation and osteogenesis of hTERT-hMSCs or primary hMSCs in porous polylactic glycolic acid (PLGA) scaffolds is described here. A cell suspension of hTERT-hMSCs or primary hMSCs (5 × 10 5 cells/250 μL) was seeded and then cultured for 12 days in porous PLGA scaffolds (10 mm in diameter, 3 mm in height) under both static and perfusion culture systems. The seeding effi-ciency, proliferation, distribution and viability, and osteogenesis of cells in scaffolds were evaluated. The perfusion method generated higher scaffold cellularity and proliferation of cells in scaffolds, and hTERT-hMSCs showed the higher proliferation potential than primary hMSCs. Results from fluorescein diacetate (FDA) staining and scanning electron microscopy (SEM) demonstrated homogeneous seeding, proliferation, and viability of hTERT-hMSCs throughout the scaffolds in the perfusion culture system. On the contrary, the static culture yielded polarized proliferation favoring the outer and upper scaffold surfaces, and resulted in decreasing of cells in the central section of the scaffolds. A flow rate of 0.5 mL/min had an effect on osteogenic differentiation of cells in scaffolds. However, the osteogenic medium promoted the osteogenic efficiency of cells. Scaffolds with hTERT-hMSCs had the higher osteogenesis than scaffolds with primary hMSCs. Thus, these results suggest that the flow condition not only allow a better seeding efficiency and homogeneity but also facilitate uniform proliferation and osteogenic differentiation of hTERT-hMSCs in scaffolds. hTERT-hMSCs could be used as stem cell candidates for bone tissue engineering experiments.